CN109897103B

CN109897103B - Stable heterodimeric antibody design with mutations in the Fc domain

Info

Publication number: CN109897103B
Application number: CN201811302754.7A
Authority: CN
Inventors: T·斯普雷特冯克罗伊登斯泰因; S·B·迪克西特; E·埃斯科巴-卡布雷拉; P·I·拉里奥; D·K·Y·潘
Original assignee: Enzymatic British Columbia Ltd
Current assignee: Enzymatic British Columbia Ltd
Priority date: 2011-11-04
Filing date: 2012-11-02
Publication date: 2024-05-17
Anticipated expiration: 2032-11-02
Also published as: JP6326371B2; JP2014533243A; CA2854233A1; US20180016347A1; US20180030150A1; EP2773671B1; HUE056462T2; EP2773671A4; BR112014010580A2; AU2012332021A8; US20150284470A1; KR20140092370A; BR112014010580B1; AU2017245451B9; PT2773671T; AU2017245451B2; AU2017245451A1; RS62689B1; RU2014121832A; AU2012332021B8

Abstract

The scaffolds provided by the present invention have heavy chains that are asymmetric in the individual domains (e.g., CH2 and CH 3) to achieve selectivity between various Fc receptors that are involved in modulating effector functions over native homodimer (symmetric) Fc molecules, and increased stability and purity of the resulting variant Fc homodimers. These new molecules comprise complexes of heterogeneous components designed to alter the natural way antibodies function and find use in therapeutic agents.

Description

Stable heterodimeric antibody design with mutations in the Fc domain

The present application is a divisional application of international application PCT/CA2012/050780 to chinese patent application (application number 201280057691.8, application day 2012, 11/2/2012, entitled "design of stable heterodimeric antibodies with mutations in Fc domain").

Introduction to the invention

Cross Reference to Related Applications

The application is based on U.S. provisional patent application No. 61/556,090 filed on 4 th month 11 of 2011, 35 th act of the United states code; U.S. provisional patent application Ser. No. 61/557,262, filed 11/8/2011; and U.S. provisional patent application Ser. No. 61/645,547, filed 5/10/2012, each of which is incorporated herein by reference in its entirety.

Technical Field

The present invention generally provides polypeptide heterodimers, compositions thereof, and methods of making and using the polypeptide heterodimers. More specifically, the invention relates to thermostable multispecific antibodies, including bispecific antibodies, comprising a heterodimeric Fc domain.

Background

Bispecific therapeutic agents are antibody-based molecules that can bind two separate and distinct targets or different epitopes of the same antigen simultaneously. Bispecific antibodies consist of immunoglobulin domain-based entities and attempt to mimic the components of antibody molecules structurally and functionally. One use of bispecific antibodies is to redirect cytotoxic immune effector cells, for example by Antibody Dependent Cellular Cytotoxicity (ADCC) to enhance killing of tumor cells. In this context, one arm of the bispecific antibody binds to an antigen on a tumor cell, while the other arm binds to a determinant expressed on an effector cell. By cross-linking the tumor and effector cells, the bispecific antibody not only introduces effector cells around the tumor cells, but also simultaneously causes activation thereof, resulting in an effective killing of the tumor cells. Bispecific antibodies have been used to enrich tumor tissue with chemical or radiation therapeutic agents to minimize deleterious effects on normal tissue. In this case, one arm of the bispecific antibody binds to an antigen expressed on the targeted cell to be destroyed, while the other arm delivers a chemotherapeutic drug, radioisotope or toxin. Beyond dual specificity, there is a need for protein therapeutics that achieve their efficacy by targeting multiple forms simultaneously. Such complex and novel biological effects can be obtained with protein therapeutics by engineering multi-target binding and multifunctional aspects in proteins.

Architecture is provided to fuse other functional warhead (war-head) or target protein binding domains in order to design a robust scaffold for these multifunctional and multi-target binding therapeutics. Desirably, the stent not only provides architecture but also provides a number of other therapeutically relevant and valuable features to the designed therapeutic agent. One of the major obstacles to the general development of antibody-based bispecific and multifunctional therapeutics is the difficulty in producing materials of sufficient quality and quantity for preclinical and clinical studies. There remains a need in the art for polypeptide constructs comprising a single variable domain as a protein binding domain linked to a variant Fc region, which variant Fc comprises a CH3 domain that has been modified to select a heterodimer with increased stability and purity.

Summary of The Invention

According to one aspect of the invention there is provided an isolated heterodimeric Fc construct comprising a modified heterodimeric CH3 domain, the modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides comprises an amino acid modification of K392J, wherein J is selected from L, I or an amino acid having a side chain volume that is not substantially greater than K; wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 74 ℃ and a purity of at least 95%; and wherein at least one amino acid modification is not a modification of an amino acid at the interface between the first and the second CH3 domain polypeptides. In certain embodiments are heterodimeric Fc constructs described herein comprising at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments are isolated heterodimeric Fc constructs described herein comprising at least one T350V modification. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a Tm of about 77 ℃ or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80 ℃ or greater. In certain embodiments, there is provided an isolated heterodimeric Fc construct described herein, wherein the at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of L351, F405, and Y407. In some embodiments are isolated heterodimeric Fc constructs wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide further comprising an amino acid modification of T366. In certain embodiments are isolated heterodimeric Fc constructs described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions L351, F405, and Y407, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions T366, K392, and T394. In embodiments are isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second CH3 domain polypeptide comprises amino acid modifications T366L, K392M and T394W. In some embodiments are isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second CH3 domain polypeptide comprises amino acid modifications T366L, K392L and T394W. In another embodiment is an isolated heterodimeric Fc construct described herein, the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second CH3 domain polypeptide comprises amino acid modifications T366I, K392M and T394W. In some embodiments are isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second CH3 domain polypeptide comprises amino acid modifications T366I, K392L and T394W. In certain embodiments are isolated heterodimeric Fc constructs described herein, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position S400. In another embodiment is an isolated heterodimeric Fc construct described herein comprising modification S400Z, wherein Z is selected from the group consisting of a positively charged amino acid and a negatively charged amino acid. In some embodiments, the positively charged amino acid is lysine or arginine and the negatively charged amino acid is aspartic acid or glutamic acid. In certain embodiments are isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide comprising an amino acid modification selected from S400E and S400R. In some embodiments, there is provided an isolated heterodimeric Fc construct described herein, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position N390. In some embodiments, the modification of N390 is N390Z, wherein Z is selected from the group consisting of a positively charged amino acid and a negatively charged amino acid. In embodiments, N390Z is N390R. In certain embodiments of the isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification S400E and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification N390R. In some embodiments of the isolated heterodimeric Fc constructs described herein, each of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide, one of which comprises the amino acid modification Q347R and the other modified CH3 domain polypeptide comprises the amino acid modification K360E.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified heterodimeric CH3 domain, the modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides comprises an amino acid modification of K392J, wherein J is selected from L, I or an amino acid having a side chain volume that is not substantially greater than K; wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 74 ℃ and a purity of at least 95%; and wherein at least one amino acid modification is not a modification of an amino acid at the interface between the first and the second CH3 domain polypeptides. In certain embodiments are heterodimeric Fc constructs described herein comprising at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments are isolated heterodimeric Fc constructs described herein comprising at least one T350V modification. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a Tm of about 77 ℃ or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80 ℃ or greater. In embodiments are isolated heterodimeric Fc constructs described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of K409 and T411. In certain embodiments is an isolated heterodimeric Fc construct described herein comprising at least one of K409F, T E and T411D. In some embodiments are isolated heterodimeric Fc constructs described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of D399. In some embodiments, the amino acid modification of D399 is at least one of D399R and D399K.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified heterodimeric CH3 domain, the modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides comprises an amino acid modification of K392J, wherein J is selected from L, I or an amino acid having a side chain volume that is not substantially greater than K; wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 74 ℃ and a purity of at least 95%; and wherein at least one amino acid modification is not a modification of an amino acid at the interface between the first and the second CH3 domain polypeptides. In certain embodiments are heterodimeric Fc constructs described herein comprising at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments are isolated heterodimeric Fc constructs described herein comprising at least one T350V modification. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a Tm of about 77 ℃ or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80 ℃ or greater. In certain embodiments of the isolated heterodimeric Fc constructs described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of K409F, T E and T411D, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of Y407A, Y407I, Y V, D399R and D399K. In some embodiments is any of the isolated heterodimeric Fc constructs described herein, further comprising a first modified CH3 domain comprising one of the amino acid modifications T366V, T366I, T366A, T M and T366L; and a second modified CH3 domain comprising the amino acid modification L351Y. In some embodiments is any of the isolated heterodimeric Fc constructs described herein comprising a first modified CH3 domain comprising one of the amino acid modifications K392L or K392E; and a second modified CH3 domain comprising one of the amino acid modifications S400R or S400V.

An isolated heterodimeric Fc construct is provided comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least four amino acid mutations, wherein at least one of the first and the second modified CH3 domain polypeptides comprises a mutation selected from the group consisting of N390Z and S400Z, wherein Z is selected from the group consisting of positively charged amino acids and negatively charged amino acids, and wherein the first and the second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain having a melting temperature (Tm) of at least about 70 ℃ and a purity of at least 90%. In embodiments, an isolated heterodimeric Fc construct is provided wherein the first modified CH3 domain polypeptide comprises an amino acid modification at positions F405 and Y407 and the second modified CH3 domain polypeptide comprises an amino acid modification at position T394. In embodiments, isolated heterodimeric Fc constructs are provided, wherein the first modified CH3 domain polypeptide comprises an amino acid modification at position L351. In certain embodiments are isolated heterodimers described herein, the second modified CH3 domain polypeptide comprising a modification at least one of positions T366 and K392. In some embodiments are isolated heterodimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ and is formed at a purity of at least about 95%. In certain embodiments are isolated heterodimers described herein, and the at least one modified CH3 domain polypeptide comprises an amino acid modification of at least one of N390R, S E and S400R. In some embodiments are isolated heterodimers described herein, one of the first and second modified CH3 domain polypeptides comprising an amino acid modification at position 347 and the other modified CH3 domain polypeptide comprising an amino acid modification at position 360. In certain embodiments are isolated heterodimers described herein, at least one of the first and second modified CH3 domain polypeptides comprising an amino acid modification of T350V. In a particular embodiment is an isolated heterodimer as described herein, the first modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of L351Y, F a and Y407V; and the second modified CH3 domain polypeptide comprises at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In certain embodiments described herein are isolated heterodimers, a first modified CH3 domain polypeptide comprising amino acid modifications at positions D399 and Y407, and a second modified CH3 domain polypeptide comprising amino acid modifications at positions K409 and T411. In some embodiments are isolated heterodimers described herein, a first CH3 domain polypeptide comprises an amino acid modification at position L351, and a second modified CH3 domain polypeptide comprises amino acid modifications at positions T366 and K392. In a particular embodiment are isolated heterodimers as described herein, at least one of the first and second CH3 domain polypeptides comprising an amino acid modification of T350V. In certain embodiments are isolated heterodimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater and is formed at a purity of at least about 95%. In certain embodiments, provided are isolated heterodimeric Fc constructs described herein, the first modified CH3 domain polypeptide comprising an amino acid modification selected from the group consisting of L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A and Y407V; and the second modified CH3 domain polypeptide comprises an amino acid modification selected from the group consisting of T366V, T366I, T366L, T366M, N390D, N390 42392E, K L, K392I, K D, K E, K409F, K409W, T D and T411E.

Provided herein are isolated heterodimeric Fc constructs comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least three amino acid mutations, wherein one of the first and the second modified CH3 domain polypeptides comprises a mutation selected from T411E and T411D, and wherein the first and the second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain having a melting temperature (Tm) of at least about 70 ℃ and a purity of at least 90%. In embodiments, an isolated heterodimeric Fc construct is provided wherein the first modified CH3 domain polypeptide comprises an amino acid modification at positions F405 and Y407 and the second modified CH3 domain polypeptide comprises an amino acid modification at position T394. In embodiments, isolated heterodimeric Fc constructs are provided, with the first modified CH3 domain polypeptide comprising an amino acid modification at position L351. In certain embodiments are isolated heterodimers described herein, the second modified CH3 domain polypeptide comprising a modification at least one of positions T366 and K392. In some embodiments are isolated heterodimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ and is formed at a purity of at least about 95%. In certain embodiments are isolated heterodimers described herein, and the at least one modified CH3 domain polypeptide comprises an amino acid modification of at least one of N390R, S E and S400R. In some embodiments are isolated heterodimers described herein, one of the first and second modified CH3 domain polypeptides comprising an amino acid modification at position 347 and the other modified CH3 domain polypeptide comprising an amino acid modification at position 360. In certain embodiments are isolated heterodimers described herein, at least one of the first and second modified CH3 domain polypeptides comprising an amino acid modification of T350V. In a particular embodiment is an isolated heterodimer as described herein, the first modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of L351Y, F a and Y407V; and the second modified CH3 domain polypeptide comprises at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In certain embodiments described herein are isolated heterodimers, a first modified CH3 domain polypeptide comprising amino acid modifications at positions D399 and Y407, and a second modified CH3 domain polypeptide comprising amino acid modifications at positions K409 and T411. In some embodiments are isolated heterodimers described herein, a first CH3 domain polypeptide comprises an amino acid modification at position L351, and a second modified CH3 domain polypeptide comprises amino acid modifications at positions T366 and K392. In a particular embodiment are isolated heterodimers as described herein, at least one of the first and second CH3 domain polypeptides comprising an amino acid modification of T350V. In certain embodiments are isolated heterodimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater and is formed at a purity of at least about 95%. In certain embodiments, provided are isolated heterodimeric Fc constructs described herein, the first modified CH3 domain polypeptide comprising an amino acid modification selected from the group consisting of L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A and Y407V; and the second modified CH3 domain polypeptide comprises an amino acid modification selected from the group consisting of T366V, T366I, T366L, T366M, N390D, N390 42392E, K L, K392I, K D, K E, K409F, K409W, T D and T411E.

Provided herein are isolated heterodimeric Fc constructs comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and the second modified CH3 domain polypeptide comprises the amino acid modifications T366I, K392M and T394W.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366I, K392L and T394W.

In a certain aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366L, K392M and T394W.

In some aspects, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366L, K392L and T394W.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modifications T350V, L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392L and T394W.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modification T350V, L351Y, S400R, F405A, Y V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392M and T394W.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modification T350V, L351Y, S400E, F405A, Y V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, N390R, K392M and T394W.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modification T350V, L351Y, F405A, Y V; and a second modified CH3 domain polypeptide comprising amino acid modifications T350V, T366L, K392L and T394W.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications T366V, K, 392L, K409F and T411E; and a second modified CH3 domain polypeptide comprising the amino acid modifications L351Y, D399R and Y407A.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising the amino acid modifications T366V, K392LE K409F and T411E; and a second modified CH3 domain polypeptide comprising the amino acid modifications L351Y, D399R, S R and Y407A.

According to one aspect of the invention, there is provided an isolated heterodimer comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain comprising an amino acid mutation that facilitates heterodimer formation, wherein the heterodimeric Fc region further comprises a variant CH2 domain comprising at least one asymmetric amino acid modification that facilitates selective binding of an fcγ receptor. In one embodiment, the variant CH2 domain selectively binds to fcγiiia receptor as compared to the wild type CH2 domain. In one embodiment, the modified CH3 domain has a melting temperature (Tm) of about 70 ℃ or greater. In certain embodiments, the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃. In some embodiments, the modified CH3 domain has a melting temperature (Tm) of at least about 80 ℃.

In another aspect, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a modified CH3 domain comprising an amino acid mutation, wherein the modified CH3 domain has a melting temperature (Tm) of about 70 ℃ or greater, and wherein the modified CH3 domain results in the formation of a heterodimeric Fc region having one or more amino acid mutations compared to a CH3 domain not comprising an amino acid mutation. In one embodiment, the heterodimeric Fc region does not comprise additional disulfide bonds in the CH3 domain relative to the wild-type Fc region. In alternative embodiments, the heterodimeric Fc region comprises at least one additional disulfide bond in the modified CH3 domain relative to the wild-type Fc region, provided that the melting temperature (Tm) of about 70 ℃ or greater is in the absence of the additional disulfide bond. In another embodiment, the heterodimeric Fc region comprises at least one additional disulfide bond in the modified CH3 domain relative to the wild-type Fc region, and wherein the modified CH3 domain has a melting temperature (Tm) of about 77.5 ℃ or greater.

In one embodiment, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a modified CH3 domain comprising an amino acid mutation, wherein the modified CH3 domain has a melting temperature (Tm) of about 70 ℃ or greater and the heterodimeric Fc region is formed at a purity of greater than about 90% or the heterodimeric Fc region is formed at a purity of greater than about 95% or greater or the heterodimeric Fc region is formed at a purity of about 98% or greater.

Also provided in certain embodiments are isolated heterodimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain comprising one or more amino acid mutations that result in the formation of a heterodimeric Fc region having one or more amino acid mutations compared to a CH3 domain not comprising the one or more amino acid mutations, wherein the modified CH3 domain has a melting temperature (Tm) of about 70 ℃ or greater or a Tm of about 71 ℃ or greater or a Tm of about 74 ℃ or greater. In another embodiment, the heterodimeric Fc region is formed in a solution having a purity of about 98% or greater and a Tm of about 73 ℃ or wherein the heterodimeric Fc region is formed at a purity of about 90% or greater and a Tm of about 75 ℃.

In certain embodiments, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first and a second CH3 domain polypeptide, wherein at least one of the first and second CH3 domain polypeptides comprises the amino acid modification T350V. In certain embodiments, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising an amino acid modification T350V and a second CH3 domain polypeptide also comprising an amino acid modification T350V. In certain embodiments, provided are isolated heterodimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising an amino acid modification at positions F405 and Y407 and a second CH3 domain polypeptide comprising an amino acid modification at position T394. The first CH3 domain polypeptide comprises amino acid modifications at positions D399 and Y407 and the second CH3 domain polypeptide comprises amino acid modifications at positions K409 and T411. In certain embodiments, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications L351Y and Y407A and a second CH3 domain polypeptide comprises amino acid modifications T366A and K409F. In one aspect, the first CH3 domain polypeptide or the second CH3 domain polypeptide comprises another amino acid modification at position T411, D399, S400, F405, N390, or K392. The amino acid modification at position T411 is selected from T411N, T411R, T411Q, T411K, T D, T E or T411W. The amino acid modification at position D399 is selected from D399R, D399W, D399Y or D399K. The amino acid modification at position S400 is selected from S400E, S400D, S R or S400K. The amino acid modification at position F405 is selected from F405I, F405M, F405T, F405S, F V or F405W. The amino acid modification at position N390 is selected from N390R, N K or N390D. The amino acid modification at position K392 is selected from K392V, K392M, K392R, K L, K392F or K392E.

In certain embodiments, provided are isolated heterodimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications T350V and L351Y and a second CH3 domain polypeptide also comprising amino acid modifications T350V and L351Y.

In another embodiment, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising amino acid modification Y407A and a second CH3 domain polypeptide comprising amino acid modifications T366A and K409F. In one aspect, the first CH3 domain polypeptide or the second CH3 domain polypeptide further comprises the amino acid modifications K392E, T411E, D399R and S400R. In another aspect, the first CH3 domain polypeptide comprises amino acid modifications D399R, S R and Y407A and the second CH3 domain polypeptide comprises amino acid modifications T366A, K409F, K392E and T411E. In another embodiment the modified CH3 domain has a melting temperature (Tm) of about 74 ℃ or greater and the heterodimer has a purity of about 95% or greater.

In another embodiment, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising an amino acid modification at position L351 and an amino acid modification Y407A and a second CH3 domain polypeptide comprising an amino acid modification at position T366 and an amino acid modification K409F. In one aspect, the amino acid modification at position L351 is selected from L351Y, L351I, L351D, L351R or L351F. In another aspect, the amino acid modification at position Y407 is selected from Y407A, Y407V or Y407S. In yet another aspect, the amino acid modification at position T366 is selected from T366A, T366I, T366L, T366M, T366Y, T366S, T366C, T366V or T366W. In one embodiment, the modified CH3 domain has a melting temperature (Tm) of about 75 ℃ or greater and the heterodimer has a purity of about 90% or greater.

In another embodiment, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising an amino acid modification at position F405 and amino acid modifications L351Y and Y407V and a second CH3 domain polypeptide comprising amino acid modification T394W. In one aspect, the first CH3 domain polypeptide or the second CH3 domain polypeptide comprises an amino acid modification at position K392, T411, T366, L368 or S400. The amino acid modification at position F405 is F405A, F405I, F405M, F405T, F405S, F405V or F405W. The amino acid modification at position K392 is K392V, K392M, K392R, K L, K392F or K392E. The amino acid modification at position T411 is T411N, T411R, T411Q, T411K, T D, T E or T411W. The amino acid modification at position S400 is S400E, S400D, S R or S400K. The amino acid modification at position T366 is T366A, T366I, T366L, T366M, T366Y, T366S, T366C, T366V or T366W. Amino acid modifications at position L368 are L368D, L368R, L368T, L M, L368V, L368F, L S and L368A.

In another embodiment, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V and a second CH3 domain polypeptide comprising amino acid modification T394W. In one aspect, the second CH3 domain polypeptide comprises amino acid modification T366L or T366I.

In yet another embodiment, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising at least one of the amino acid modifications Y349C, F a and Y407V and a second CH3 domain polypeptide comprising the amino acid modifications T366I, K392M and T394W.

In certain embodiments, provided are isolated heterodimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V and a second CH3 domain polypeptide comprising amino acid modifications K392M and T394W, and one of T366L and T366I.

In another embodiment, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications F405A and Y407V and a second CH3 domain polypeptide comprising amino acid modifications T366L and T394W.

In another embodiment, an isolated heterodimer comprising a heterodimeric Fc region is provided, wherein the heterodimeric Fc region comprises a first CH3 domain polypeptide comprising amino acid modifications F405A and Y407V and a second CH3 domain polypeptide comprising amino acid modifications T366I and T394W.

Bispecific or multispecific antibodies are provided in certain embodiments of heterodimers.

In another embodiment, a composition comprising a heterodimer of the invention and a pharmaceutically acceptable carrier is provided.

In another embodiment, a host cell comprising a nucleic acid encoding a heterodimer of the invention is provided.

In certain embodiments, heterodimers are provided, wherein the heterodimer comprises at least one therapeutic antibody. In one aspect, the therapeutic antibody is selected from the group consisting of: abamelizumab (abagovomab), adalimumab (adalimumab), alemtuzumab, ologegrel (aurograb), bapiduzumab, bazeb, basilizumab (belimumab), bevacizumab, briakinumab, kanaduzumab (canakinumab), cetuximab (catumaxomab), sakuizumab (certolizumab pegol), cetuximab, daclizumab, destuzumab (denosumab), efalizumab, gancicximab (galiximab), gemtuzumab, ozagrimoxin, golimumab (golimumab), temozolomab, infliximab, ipilimumab, lu Xishan, meperimumab, motuzumab, mycograb, natalizumab, nimuzumab, orelizumab (ocrelizumab), fabizumab (ofatumab), oxuzumab, palitumomab, panitumomab, ceritumomab, silab, ceritumomab, 68zuab, and other anti-tulizumab.

In another embodiment of the heterodimer of the invention, a method of treating cancer in a patient having cancer characterized by a cancer antigen is provided, the method comprising administering to the patient a therapeutically effective amount of the heterodimer.

In another embodiment of the heterodimer of the invention, a method of treating an immune disorder in a patient having an immune disorder characterized by an immune antigen is provided, the method comprising administering to the patient a therapeutically effective amount of the heterodimer.

In certain embodiments, the modified Fc region used in the heterodimer constructs described herein comprises a type G immunoglobulin, e.g., an immunoglobulin defined as a type 2 immunoglobulin (IgG 2) or a type 3 immunoglobulin (IgG 3). In some embodiments, the modified Fc region used in the heterodimer constructs described herein comprises immunoglobulin M or IgM. In some embodiments, the modified Fc region used in the heterodimer constructs described herein comprises immunoglobulin a or IgA. In some embodiments, the modified Fc region used in the heterodimer constructs described herein comprises immunoglobulin D or IgD. In some embodiments, the modified Fc region used in the heterodimer constructs described herein comprises immunoglobulin E or IgE. In certain embodiments, the modified Fc region used in the heterodimer constructs described herein comprises all classes of immunoglobulin G isotypes, e.g., immunoglobulins defined as class 1 (IgG 1), class 2 (IgG 2), class 3 (IgG 3), or class 4 (IgG 4) immunoglobulins.

Brief Description of Drawings

FIG. 1 is a schematic 3-D structure of a wild-type antibody showing CH3 (top), CH2 (middle) and acceptor regions. The dashed rectangle on the left hand side extends to the right hand side, which shows two regions of the target area of CH3, region 1 and region 2;

FIG. 2 is a graphical 3-D representation showing the wild-type residue at position 368;

FIG. 3 is a graphical 3-D representation of region 1 showing position 368 of the mutation;

FIG. 4 is a graphical 3-D representation showing additional mutations in region 2;

FIG. 5 is a table of on-chip calculated values for collision scores, interface area differences, packaging differences, electrostatic energy differences, and overall "affinity scores" for the first three variants AZ1, AZ2, and AZ 3;

FIG. 6 is a graphical 3-D image showing variants AZ2 and AZ3 "built on" variant AZ 1;

FIG. 7 shows a graphical 3-D representation of AZ2 and AZ3 variants;

fig. 8 shows a table as in fig. 5 but for AZ1, AZ2 and AZ3 heterodimers and potential homodimers. Affinity scores for homodimers were not shown, as they were irrelevant;

FIG. 9 is a graphical representation of 3-D representations of wild-type (left) and mutated AZ4 (right);

FIG. 10 shows a table of calculated on-chip values for the AZ4 heterodimer and potential homodimers as in FIG. 5;

FIG. 11 is a graphical representation of the CH3 variants AZ5 (left) and AZ6 (right);

FIG. 12 is a table as described in FIG. 5 and showing chip-on-computer data for AZ4, AZ5 and AZ 6;

FIG. 13 is a graphical 3-D representation of antibodies on the left with the potential for binding characteristics at the receptor region using a heterodimerization approach;

FIG. 14 is a schematic representation of an IgG molecule;

Figure 15 shows multiple sequence alignments of fcγ receptors. Genebank/Uniprot sequence ID： FcγRIIA(sp P12318)、FcγRIIB(sp P31994)、FcγRIIC(gi 126116592)、 FcγRIIIA(sp P08637)、FcγRIIIB(sp O75015);

FIG. 16 is a graphical representation of the crystal structure of Fc-FcgammaRIIIb complexes [ PDB ID:1T83, radaev & Sun ]. The 1:1 complex of Fc and fcγreceptor is observed to have asymmetric contact between the two chains of Fc and fcγr;

figure 17 shows a graphical representation of multifunctional molecules based on asymmetric Fc scaffolds formed from the heterodimeric variants described herein: an asymmetric Fc scaffold and an asymmetric Fc monomer IgG arm;

Fig. 18 shows a graphical representation of multifunctional molecules based on asymmetric Fc scaffolds formed from the heterodimeric variants described herein: asymmetric Fc monospecific IgG arms and asymmetric Fc bispecific IgG arms (sharing a light chain);

Figure 19 shows a graphical representation of multifunctional molecules based on asymmetric Fc scaffolds formed from the heterodimeric variants described herein. Asymmetric Fc bispecific IgG arms and functional molecules such as toxins;

Figure 20 shows a multifunctional molecule based on an asymmetric Fc scaffold formed from the heterodimeric variants described herein: an asymmetric Fc single scFv arm and an asymmetric Fc bispecific scFv arm;

Figure 21 shows alternative multifunctional molecules based on asymmetric Fc scaffolds formed from the heterodimeric variants described herein: asymmetric Fc trispecific scFv arms and asymmetric Fc tetraspecific scFv arms.

Figure 22 shows that asymmetric design mutations on one side of Fc for better fcγr selectivity introduce a generating side for fcγr interactions and a non-generating side with wild-type-like interactions. Mutations on the non-producing side of Fc can be introduced to block interactions with FcR and Fc favors polarity so that interactions only occur on the producing side.

FIG. 23 shows the amino acid sequence of a parent wild-type heavy chain human IgG1 sequence.

Fig. 24 shows an iterative process for Fc heterodimer design, combining positive and negative design strategies as detailed below.

FIGS. 25A-25C show in vitro assays for determining heterodimer purity. The assay is based on a full length monospecific antibody scaffold with two Fc heavy chains of different molecular weight; heavy chain a has a C-terminal His tag (His) and heavy chain B has a C-terminal cleavable mRFP tag (RFP). Two heavy chains a (His) and B (RFP) were expressed in different relative ratios together with fixed amounts of light chain, yielding 3 possible dimer species with different molecular weights: a) Homodimeric chain a (His)/chain a (His) (about 150 kDa); b) Heterodimeric chain a (His)/chain B (RFP) (about 175 kDa); c) Homodimeric chain B (RFP)/chain B (RFP) (about 200 kDa). After expression, the proportion of heterodimers compared to the two homodimers was determined by non-reducing SDS-PAGE, which allowed separation of 3 dimer species by molecular weight, as described in example 2. SDS-PAGE gels were stained with Coomassie brilliant blue. Fig. 25A: the variant tested was WT chain a (His) alone; WT chain B (RFP) only; WT strand a (His) plus strand B (RFP); control 1 chain a (His) plus chain B (RFP) with >95% of the reported heterodimeric purity. The composition of the dimer bands was verified by Western blotting with antibodies directed against IgG-Fc (anti-Fc), mRFP Tag (anti-mRFP) and HisTag (anti-His) as described above. SDS-PAGE shows a single band for His/His homodimers, a double band for His/RFP heterodimers and multiple bands for RFP homodimers. The multiple bands are mRFP-labeled artifacts and have been shown not to affect the physical properties of Fc heterodimers. Fig. 25B: SDS-PAGE assays were validated against published Fc heterodimer variant controls 1-4, see Table A. Variants were expressed in different relative proportions of chain a (His) compared to chain B (RFP): specifically, the ratio 1:3 is equal to the LC, hc_his, hc_mrfp ratio of 25%, 10%, 65%, respectively; ratio 1:1 equals 25%, 20%, 55% LC, hc_his, hc_mrfp ratio and ratio 3:1 equals 25%, 40%, 35% LC, hc_his, hc_mrfp ratio (apparent 1:1 expression of chain a (His) to chain B (RFP) has been determined to be near 20%/55% (His/RFP) for WT Fc). FIG. 25C shows a non-reducing SDS-PAGE assay to determine heterodimeric purity of scaffold 1 variants. Fc variants were expressed in different relative proportions of chain a (His) compared to chain B (RFP) and analyzed by non-reducing SDS-PAGE as described in fig. 2. Specifically, the ratio 1:3 is equal to the LC, hc_his, hc_mrfp ratio of 25%, 10%, 65%, respectively; ratio 1:1 equals 25%, 20%, 55% LC, hc_his, hc_mrfp ratio and ratio 3:1 equals 25%, 40%, 35% LC, hc_his, hc_mrfp ratio (apparent 1:1 expression of chain a (His) to chain B (RFP) has been determined to be near 20%/55% (His/RFP) for WT Fc).

FIGS. 26A-26B show heterodimeric variants expressing Fc at a specific ratio of chain A (His) compared to chain B (RFP) (see Table 2), purified by protein A affinity chromatography and analyzed by non-reducing SDS-PAGE as described in FIGS. 25A-25C. FIG. 26A illustrates the sorting of heterodimers based on purity observed by visual inspection of SDS-PAGE results. For comparison, an equal amount of protein a purified product was loaded onto the gel. The definition of purity based on non-reducing SDS-PAGE has been verified by LC/MS for selected variants (see FIG. 28). FIG. 26B provides exemplary SDS-PAGE results of selected protein A purified heterodimer variants (AZ 94, AZ86, AZ70, AZ33, and AZ 34).

FIGS. 27A-27B illustrate DSC analysis used to determine the melting temperature of a heterodimerized CH3-CH3 domain formed from the heterodimeric variants described herein. Two independent methods are used to determine the melting temperature. Figure 27A provides a thermogram fitted to 4 independent non-2 state-transitions (non-2-state-transitions) and optimized to produce CH2 and Fab transition values close to the reported literature values for Herceptin of about 72 ℃ (CH 2) and about 82 ℃ (Fab). Figure 27B shows normalized and baseline corrected thermograms of the heterodimer variant subtracted from WT to yield positive and negative difference peaks for CH3 conversion only.

Fig. 28 illustrates LC/MS analysis of example variant AZ70 as described in example 2. The expected (calculated average) quality of glycosylated heterodimers and homodimers is noted. The region consistent with heterodimer mass contains major peaks corresponding to the loss of glycine (-57 Da) and the addition of 1 or 2 hexoses (+162 Da and +324Da, respectively). Heterodimer purity is classified as >90% if there is no distinct peak corresponding to either homodimer.

29A-29D show analysis of initial negative design variants and independent positive design optimizations on a computer chip: scaffold 1-hydrophobic core. WT is shown (fig. 29A); AZ6 (fig. 29B); AZ33 (fig. 29C); and the CH3 interface of AZ19 (fig. 29D). Comparison of the comprehensive analysis and variants on the computer chip as described in the detailed description with WT shows that one of the reasons for the lower stability of the initial AZ33 heterodimer than WT stability is the loss of core interactions/packaging of Y407 and T366. The initial AZ33 shows a non-optimal packing at the hydrophobic core as shown in fig. 29B, indicating that optimization of this region, especially at position T366, can improve the stability of AZ 33. This is illustrated in fig. 29C and 29D by T366I and T366L. Experimental data is relevant to this structural analysis and shows that T366L gives the greatest improvement in Tm. See, example 5.

FIG. 30 shows analysis of initial negative design variants and independent positive design optimizations on a computer chip: the scaffold 1-stable 399-400 loop configuration illustrates the utility and importance of the conformational kinetic analysis exemplified in the initial scaffold 1 variant AZ 8. The mutagenized structure (near the framework configuration of WT) was overlaid on a computer chip with a representative structure of 50ns molecular dynamics simulation analysis. The figure highlights the larger conformational difference of AZ8 variants compared to the loop region D399-S400 of WT, which in turn exposes the hydrophobic core to solvent and causes a decrease in stability of AZ8 heterodimers.

FIGS. 31A-31C show analysis of initial negative design variants and independent positive design optimizations on a computer chip: the scaffold 1-stabilizes 399-400 loop configuration, illustrating how information from on-chip analysis and MD simulation is used for the positive design strategy. As shown in fig. 30, one of the reasons for AZ8 stability below WT stability is the reduced interaction of loops 399-400 with 409, mainly due to the loss of F405 packaging interaction (see comparison of fig. 31A (WT) versus fig. 31B (AZ 8)). One of the positive design strategies is to optimize the hydrophobic packaging of the region to stabilize the 399-400 loop conformation. This was achieved by the K392M mutation shown in fig. 31C. Fig. 31C represents the heterodimer AZ33, which has a Tm of 74 °, in contrast to the original negative design variant AZ8, which has a Tm of 68 °.

FIGS. 32A-32B illustrate the kinetics of Fc molecules observed using principal component analysis of molecular kinetic trajectories. Fig. 32A shows a skeleton trace of an Fc structure as a reference. Figures 32B and C represent the kinetic overlap observed along the top 2 primary modes of motion in the Fc structure. The CH2 domains of chains a and B exhibit significant on/off movement relative to each other, while the CH3 domains are relatively rigid. Mutations at the CH3 interface affect the relative flexibility and dynamics of this on/off movement in the CH2 domain.

Figures 33A-33C show analysis of initial negative design variants and independent positive design optimizations on a computer chip: scaffold 2-hydrophobic packaging, illustrating the hydrophobic core packaging of two scaffold 2 variants compared to WT. WT Fc (fig. 33A); AZ63 (fig. 33B); and AZ70 (fig. 33C). Comprehensive analysis on the computer chip of the initial scaffold 2 variant showed that loss of core WT interaction of Y407-T366 was one of the reasons for the lower stability of the initial scaffold 2 variant than WT stability. The loss of Y407-T366 was partially compensated by mutation K409F, but as shown in fig. 33B, in particular the T366A mutation left a cavity in the hydrophobic core, which destabilizes the variant compared to WT. As shown in Fc variant AZ70 in fig. 33C, targeting the hydrophobic core by the additional mutation T366v_l351Y proved successful; AZ70 has an experimentally determined Tm of 75.5 ℃. See, table 4 and example 6.

Figures 34A-34C show analysis of initial negative design variants and independent positive design optimizations on a computer chip: scaffold 2-stabilization 399-400 loop configuration, illustrating the interaction of two scaffold 2 variants compared to loops 399-400 of WT: WT Fc (fig. 34A); AZ63 (fig. 34B); and AZ94 (fig. 34C). Comprehensive analysis on the computer chip of the initial scaffold 2 variant showed that loss of WT salt bridge K409-D399 due to mutation K409F (fig. 34A) and thus unsatisfactory D399 (fig. 34B) caused a more 'open' conformation of the 399-400 loops. This in turn results in greater solvent exposure of the hydrophobic core and further instability of the variants compared to WT. One of the strategies for stabilizing the 399-400 loops and compensating for the loss of K409-D399 interaction was to design additional salt bridges D399R-T411E and S400R-K392E as shown in figure 34C for variant AZ 94. Experimental data showed a purity of >95% and Tm at 74 ℃. See, table 4 and example 6. Furthermore, although AZ94 has a considerably higher purity and stability compared to the original scaffold 2 variant (purity <90%, tm 71 ℃), the hydrophobic core mutation of AZ94 is less preferred than the "optimal" hydrophobic core mutation identified in the AZ70 variant (fig. 33). Since the mutation of the hydrophobic core in AZ70 (t366 v_l351Y) is distal to the salt bridge mutation of AZ94 at loops 399-400, the combination of AZ70 amino acid mutation and additional AZ94 mutation is expected to have a higher melting temperature than either AZ70 or AZ 94. The combination can be tested as described in examples 1-4.

FIG. 35 shows the binding constants of homodimerized IgG1 Fc, heterodimerized variant het1 (control 1) A:Y349C_T366S_L368A_Y407V/B S354C_T366W and het2 (control 4) A:K409D_K392D/B D399K_D356K for binding to six Fcgamma receptors (Ka (M ^-1)). Heterodimeric Fc variants tend to show slight alterations in binding to fcγ receptors compared to wild-type IgG1 Fc. See, example 7.

FIG. 36A shows the relative binding strength of wild-type IgG1 Fc and its various homodimeric and asymmetric mutant forms to IIbF, IIBY and IIaR receptors based on wild-type binding strength as a reference. (homoFc+S267D) refers to the binding strength of homodimerized Fc to the S267D mutation on both chains. (hetfc+asym S267D) refers to the binding strength of homodimerized Fc to the S267D mutation introduced in one of the two chains in Fc. The average value of binding strength obtained by introducing mutations on either of the two Fc chains is reported. Introducing this mutation on one strand reduces the binding strength to about half the strength observed for the same mutation in the homodimeric form. (hetfc+asym S267d+asym E269K) refers to the binding strength of homodimerized Fc to one of the two Fc chains in which the S267D and E269K mutations were introduced in an asymmetric manner. The E269K mutation blocked the FcgR interaction with one face of the Fc and was able to reduce the binding strength by about half that observed for the asymmetric S267D variant (hetfc+s267D) itself. Het Fc here consists of the CH3 mutation indicated in figure 35 for variant Het2 (control 4).

FIG. 36B shows the binding constants of various Fc and variants thereof to various FcgRIIa, fcgRIIb and FcgRIIIa allotypes (Ka (M ^-1)). Ka of the wild-type IgG1 Fc for the various Fcg receptors is shown as a horizontally shaded bar. The bar with vertical shading (homodimer base 2) represents the heterodimer Fc with the Ka of the mutation S239D/D265S/I332E/S298A. The columns with oblique shading represent the asymmetric mutations A: S239D/D265S/I332E/E269K and B: ka of S239D/D265S/S298A in the homodimer Fc and CH2 domains. The introduction of asymmetric mutations enables increased selectivity between IIIa and IIa/IIb receptors. The heterodimeric Fc here consisted of the CH3 mutation indicated in fig. 35 for variant het2 (control 4).

FIG. 36C shows the binding constants of three other variants in the CH2 domain of wild-type IgG1 and Fc regions involving homodimeric or asymmetric mutations (Ka (M ^-1)). Ka of wild-type Fc is shown as a grid-hatched bar. The Fc variant is shown in a bar in oblique mode with the Ka of the basic mutation S239D/K326E/A330L/I332E/S298A introduced in homodimeric manner (homodimeric basis 1) on both chains of the Fc. Relevant mutations are introduced in asymmetric fashion in chains a and B of the heterodimeric Fc (heterodimeric basis 1) and are shown in horizontal lines. The columns with vertical hatching represent asymmetric variants (heterologous basis 1+pd) comprising the E269K mutation. The heterodimeric Fc here consisted of the CH3 mutation indicated in fig. 35 for variant het2 (control 4).

Fig. 37-table 6 are lists of variant CH3 domains based on the third design stage as described for scaffold 1 in example 5.

FIGS. 38-Table 7 are lists of modified CH3 domains based on the third design stage as described for scaffold 2 in example 6.

FIGS. 39A-39B illustrate purity determination of variants without any C-terminal labeling using LC/MS. FIG. 39A shows the LC/MS spectrum (AZ 162: L351Y_F405A_Y407V/T366L_K392L_T 394W) of a representative variant. As described in the examples, variants were expressed by transient co-expression using 3 different ratios of heavy chain-a to heavy chain-B of 1:1.5 (AZ 133-1), 1:1 (AZ 133-2) and 1.5:1 (AZ 133-3). The samples were purified and deglycosylated with Endo S at 37 ℃ for 1 hour. Samples were injected into Poros R2 column and eluted in a gradient with 20-90% acn,0.2% fa for 3 min prior to MS analysis. The peaks of the LC column were analyzed with an LTQ-Orbitrap XL mass spectrometer (cone aperture voltage: 50V' tube lens: 215V; FT resolution: 7,500) and integrated with software Promass to produce a molecular weight distribution. FIG. 39B shows the LC/MS spectrum of the control 2 sample, which represents the nodular pore variant. Variants were expressed by transient co-expression using 3 different ratios of heavy chain-A to heavy chain-B of 1:1.5 (control 2-1), 1:1 (control 2-2) and 1.5:1 (control 2-3), as described in the examples. The samples were purified and deglycosylated with Endo S at 37 ℃ for 1 hour. Samples were injected into Poros R2 column and eluted in a gradient with 20-90% acn,0.2% fa for 3 min prior to MS analysis. The peaks of the LC column were analyzed with an LTQ-Orbitrap XL mass spectrometer (cone aperture voltage: 50V' tube lens: 215V; FT resolution: 7,500) and integrated with software Promass to produce molecular weight distribution.

FIGS. 40A-40B demonstrate bispecific binding using Fc heterodimers anti-HER 2 and anti-HER 3 scFv fused to the N-terminus of chain-A and chain-B of the Fc heterodimer. The variant bispecific HER2/HER3 variants obtained and the two monovalent-monospecific HER2, HER3 variants (dark grey chain-a; light grey chain-B) are illustrated in fig. 40-a. FIG. 40-B demonstrates the test of bispecific binding.

FIG. 41 illustrates a computational model comparing wild-type IgG1 Fc and AZ 3003. The computational model of AZ3002 is identical to AZ3003 at position T350. The table summarizes the stabilizing effect of the selected heterodimer variants and T350V mutations on CH3 melting temperature. The figure shows the expression and purification of heterodimeric variants as described in example 11. DSC was performed as described in example 3 and LC/MS quantification was performed as described in example 11.

FIG. 42 illustrates a comparison of the crystal structure of the lead heterodimer and a predicted model. The mutated interface residues are highlighted in the cartoon representation (shown in the table).

FIG. 43 depicts analysis of glycosylation patterns of purified lead heterodimers.

FIG. 44 illustrates the results of forced degradation assessment of purified lead heterodimers.

FIG. 45 depicts an industry standard antibody purification process flow.

Fig. 46 depicts a summary of downstream purification evaluations of AZ3003 heterodimer variants, showing step yields and recovery (see example 15 for details). Heterodimers were prepared in 10L of transient CHO as described in detail in example 11.

Detailed description of the preferred embodiments

Provided herein are modified CH3 domains comprising specific amino acid modifications that promote heteromultimer formation. In one embodiment, the modified CH3 domain comprises a particular amino acid modification that promotes heterodimer formation (see, e.g., tables 1.1-1.3). In another embodiment, the modified CH3 domain comprises a specific amino acid modification that promotes heterodimer formation with increased stability (see, e.g., table 4, table 6, and table 7). Stability is measured as the melting temperature (Tm) of the CH3 domain, and increased stability refers to Tm of about 70 ℃ or higher. The CH3 domain forms part of the Fc region of a heteromultimer or multispecific antibody. Thus, in one embodiment, provided herein is a heteromultimer comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified or variant CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain is selected from the variants listed in table 1. In a second embodiment, a heteromultimer is provided comprising a heteromultimeric Fc region, wherein the heteromultimeric Fc region comprises a variant CH3 domain having increased stability, the modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) of about 70 ℃ or greater.

Amino acid modifications used to generate modified CH3 domains include, but are not limited to, amino acid insertions, deletions, substitutions, and rearrangements. Modifications of CH3 domains and modified CH3 domains are collectively referred to herein as "CH3 modifications", "modified CH3 domains" or "CH3 variants". In certain embodiments, these modified CH3 domains are incorporated into selected molecules. Accordingly, in one embodiment, molecules, e.g., polypeptides, such as immunoglobulins (e.g., antibodies) and other binding proteins, are provided that comprise an Fc region incorporating a modified CH3 domain (as used herein, "Fc region" and like terms include any heavy chain constant region domain comprising at least a portion of a CH3 domain). Molecules comprising an Fc region comprising a modified CH3 domain (e.g., a CH3 domain comprising one or more amino acid insertions, deletions, substitutions, or rearrangements) are referred to herein as "Fc variants", "heterodimers", or "heteromultimers". The present Fc variants comprise CH3 domains that have been asymmetrically modified to produce heterodimeric Fc variants or regions. The Fc region is made up of two heavy chain constant domain polypeptides, chain a and chain B, which can be used interchangeably, provided that each Fc region comprises one chain a and one chain B polypeptide. Amino acid modifications are introduced into CH3 in an asymmetric manner, resulting in heterodimers when two modified CH3 domains form an Fc variant (see, e.g., table 1). As used herein, an asymmetric amino acid modification is any modification in which the amino acid at a particular position on one polypeptide (e.g., "chain a") differs from the amino acid at the same position on a second polypeptide (e.g., "chain B") of a heterodimer or Fc variant. This may be the result of a modification of only one of the two amino acids or of both amino acids from the two different amino acids of chain a and chain B of the Fc variant. It will be appreciated that the modified CH3 domain comprises one or more asymmetric amino acid modifications.

The amino acid at the interface between the first and said second CH3 domain polypeptides is any amino acid on the first or second CH3 domain polypeptide that interacts with an amino acid on the other CH3 domain polypeptide (resulting in the formation of a dimerized CH3 domain). Amino acids that are not at the interface between the first and said second CH3 domain polypeptides are any amino acid on the first or second CH3 domain polypeptide that does not interact with an amino acid on the other CH3 domain polypeptide. In embodiments described herein, the amino acid modified not at the interface between the first and the second CH3 domain polypeptide is any amino acid on the first or second CH3 domain polypeptide that is not modified as described herein with an amino acid on the other CH3 domain polypeptide. For example, in certain embodiments described herein, modifications of amino acid position T350 are provided. As demonstrated by the crystal structure provided in example 12 and shown in fig. 42, T350 does not participate in the interaction between the two CH3 domain polypeptides. Any modification to T350 has been shown to have a negligible effect on CH3 dimer formation, as described by Carter et al Biochemistry 1998,37,9266. In the heterodimeric Fc constructs described herein, the modification at the T350 position was observed to have an undesirable stabilizing effect on the variant CH3 domain, although it was not directly involved in the formation of the CH3 dimer itself. For example, variants comprising at least one T350X modification (wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof) form a very stable variant CH3 domain. In some embodiments described herein are isolated heterodimeric Fc constructs described herein comprising at least one T350V modification. In certain embodiments, the first and second variant CH3 domain polypeptides comprise a T350V modification that imparts an undesirable stability to the variant CH3 domain as compared to a corresponding CH3 domain that does not comprise the modification.

Unless otherwise indicated, in this specification any concentration range, percentage range, ratio range, or integer range is to be understood to include any integer value within the range, as well as fractional values thereof (e.g., one tenth and one hundredth of an integer) as appropriate. As used herein, unless otherwise indicated, "about" refers to ± 10% of the stated range, value, sequence, or structure. It is to be understood that the terms "a" and "an" as used herein mean "one or more" of the recited components unless specified otherwise or indicated by context. The use of alternatives (e.g., "or") is to be understood as one, two, or any combination thereof of the alternatives. The terms "comprising" and "including" as used herein may be used synonymously. Furthermore, it is to be understood that individual single chain polypeptides or heterodimers derived from various combinations of the structures and substitutions described herein (e.g., modified CH3 domains) are disclosed to the same extent as if each single chain polypeptide or heterodimer were listed individually. Thus, the choice of a particular component to form a single chain polypeptide or heterodimer falls within the scope of the application.

A "first polypeptide" is any polypeptide that binds to a second polypeptide, also referred to herein as "chain A". The first and second polypeptides meet at an "interface". A "second polypeptide" is any polypeptide that binds to a first polypeptide via an "interface", also referred to herein as "chain B". "interface" includes those "contact" amino acid residues in the first polypeptide that interact with one or more "contact" amino acid residues in the interface of the second polypeptide. As used herein, the interface comprises a CH3 domain of an Fc region, which is preferably derived from an IgG antibody, most preferably a human IgG ₁ antibody.

As used herein, "isolated" heteromultimer means a heteromultimer that has been identified and isolated and/or recovered from components of its natural cell culture environment. The contaminating components of its natural environment are substances that can interfere with the diagnostic or therapeutic use of the heteromultimer, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes.

The side chain volume "substantially no greater than" the amino acid of the first amino acid is based on the side chain volume ratio from A.A.Zamyatnin, prog.Biophys.Mol.Biol.24:107-123,1972, the first amino acid having a side chain volume value of no more thanAny amino acid of (a) and (b). In certain embodiments, the volume is no more than/>, greater than the first amino acidIn some embodiments, the volume is no more than/>, greater than the first amino acidFor example, in certain embodiments described herein is a mutation of lysine (K) such as K392J, wherein J is selected from L, I or an amino acid having a side chain volume that is not substantially greater than the side chain volume of K. /(I)

Variant Fc heterodimers are generally purified to substantial homogeneity. The phrases "substantially homogeneous", "substantially homogeneous form" and "substantially homogeneous" are used to indicate that the product is substantially free of byproducts (e.g., homodimers) derived from unwanted combinations of polypeptides. By substantially homogenous in terms of purity is meant that the amount of by-products is not more than 10%, preferably less than 5%, more preferably less than 1%, most preferably less than 0.5%, wherein the percentages are by weight.

The terms understood by those skilled in the art of antibody technology each have the meaning obtained in the art unless explicitly defined differently herein. Antibodies are known to have variant regions, hinge regions and constant domains. For a review of immunoglobulin structure and function see, e.g., harlow et al, antibodies: a Laboratory Manual, chapter 14 Cold Spring Harbor Laboratory, cold Spring Harbor, 1988).

Variant Fc heterodimers were designed from wild-type homodimers by the concept of positive and negative design in the context of protein engineering by equilibrium stability versus specificity, where mutations were introduced to drive heterodimer formation beyond homodimer formation of interest when the polypeptide was expressed in cell culture conditions. The negative design strategy (Gunaskekaran K, et al Enhancing antibody Fc heterodimer formation through electrostatic steering effects:applications to bispecific molecules and monovalent IgG.JBC 285(25):19637-19646(2010)) negative design strategy maximizes adverse interactions on homodimer formation by introducing large side chains on one strand and small side chains on the other strand, such as the nodular access (Knobs-intos) strategy (Ridgway JB,Presta LG,Carter P.'Knobs-into-holes'engineering of antibody CH3domains for heavy chain heterodimerization.Protein Eng. 1996Jul;9(7):617-21;Atwell S,Ridgway JB,Wells JA,Carter P.Stable heterodimers from remodeling the domain interface of a homodimer using a phage display library.J Mol Biol.270(1):26-35(1997))), developed by Genentech, or by electrostatic engineering leading to rejection of homodimer formation (Gunaskekaran K, et al Enhancing antibody Fc heterodimer formation through electrostatic steering effects:applications to bispecific molecules and monovalent IgG.JBC 285(25):19637-19646(2010)) negative design strategy) in both examples, negative design asymmetric point mutations are introduced into wild-type CH3 domains to drive heterodimer formation.

Table a: published Fc heterodimeric antibodies

* We observed >90% purity of control 1 in our assay system, but not 100% as reported previously in the literature.

* We observed Tm above 77 ℃ for control 4 in our assay system; the Tm for this variant has not been disclosed in the literature.

NP-has not been disclosed for the Tm of control 3 and it was not tested in our assay system.

The melting temperature of wild-type IgG1 is shown to be in the range of 81-83, since the values in the literature vary depending on the assay system used, we report values of 81.5 ℃ in our assay system.

In contrast to negative designs, the general concept for engineering proteins is a positive design. In this case, amino acid modifications are introduced into the polypeptide to maximize the beneficial interactions within or between proteins. This strategy assumes that when multiple mutations are introduced that specifically stabilize the desired heterodimer while ignoring the effects on the homodimer, the net effect will be better specificity for the desired heterodimer interactions compared to homodimers, and thus higher heterodimer specificity. In the context of protein engineering, it can be appreciated that positive design strategies optimize the stability of the desired protein interactions, but rarely achieve >90% specificity (Havranek JJ&Harbury PB.Automated design of specificity in molecular recognition.Nat Struct Biol.10(1):45-52(2003);Bolon DN,Grant RA,Baker TA,Sauer RT.Specificity versus stability in computational protein design.Proc Natl Acad Sci USA.6;102(36):12724-9(2005);Huang PS,Love JJ,Mayo SL.A de novo designed protein protein interface Protein Sci.16(12):2770-4(2007)). prior to the present disclosure, were not used to design Fc heterodimers because of the greater focus on specificity input than therapeutic antibody production and development. Furthermore, favorable positive design mutations are difficult to predict. Other methods for improving stability, such as additional disulfide bonds, have been tried to improve the stability of Fc heterodimers, but the success of the improvement on the molecules is limited. (see, table a) this is likely because all engineered Fc CH3 domain disulfide bonds are solvent exposed, which results in a shorter life cycle of disulfide bonds, and thus a significant impact on the long term stability of the heterodimer-especially when the engineered CH3 domain has a Tm of less than 70 ℃ without additional disulfide bonds (as in control 4, which has a Tm of 69 ℃ without additional disulfide bonds (see control 2) -other methods of improving stability, such as disulfide bonds, can also be used with the Fc variants of the invention, provided that the inherent stability of the CH3 domain without disulfide bonds (measured as melting temperature) is 70 ℃ or higher, especially when the inherent stability of the CH3 domain without disulfide bonds (measured as melting temperature) is 72 ℃ or higher.

Thus, we disclose herein a new method for designing Fc heterodimers, which results in stable and highly specific heterodimer formation. This design approach combines negative and positive design strategies along with structural and computational model-directed protein engineering techniques. This powerful approach has allowed us to design a new combination of mutations in the IgG1 CH3 domain, where heterodimers are formed using only standard cell culture conditions, which have a purity of over 90% compared to homodimers, and the resulting heterodimers have a melting temperature of 70 ℃ or higher. In exemplary embodiments, the Fc variant heterodimer has a melting temperature of 73 ℃ or greater and a purity of greater than 98%. In other exemplary embodiments, the Fc variant heterodimer has a melting temperature of 75 ℃ or greater and a purity of greater than 90%. In certain embodiments of the heterodimeric Fc variants described herein, the Fc variant heterodimer has a melting temperature of 77 ℃ or greater and a purity of greater than 98%. In certain embodiments of the heterodimeric Fc variants described herein, the Fc variant heterodimer has a melting temperature of 78 ℃ or greater and a purity of greater than 98%. In certain embodiments of the heterodimeric Fc variants described herein, the Fc variant heterodimer has a melting temperature of 79 ℃ or greater and a purity of greater than 98%. In certain embodiments of the heterodimeric Fc variants described herein, the Fc variant heterodimer has a melting temperature of 80 ℃ or greater and a purity of greater than 98%. In certain embodiments of the heterodimeric Fc variants described herein, the Fc variant heterodimer has a melting temperature of 81 ℃ or greater and a purity of greater than 98%.

In certain embodiments, isolated heteromultimers comprising a heteromultimeric Fc region are provided, wherein the heteromultimeric Fc region comprises a modified CH3 domain having increased stability, the modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) of 70 ℃ or greater. As used herein, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 70 ℃ or higher. In certain embodiments, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 72 ℃ or greater. In certain embodiments, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 74 ℃ or higher. In certain embodiments, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 75 ℃ or greater. In certain embodiments, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 76 ℃ or greater. In certain embodiments, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 78 ℃ or higher. In certain embodiments, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 79 ℃ or higher. In certain embodiments, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 80 ℃ or greater. In certain embodiments, "increased stability" or "stable heterodimer" refers to a modified CH3 domain in heterodimer formation that has a melting temperature of about 81 ℃ or higher. Furthermore, it is understood that the term "promote heterodimer formation" refers herein to amino acid mutations in the CH3 domain that result in greater than 90% heterodimer formation compared to homodimer formation.

In further embodiments, this increased stability is in the absence of additional disulfide bonds. In particular, the increased stability is in the absence of additional disulfide bonds in the CH3 domain. In one embodiment, the modified CH3 domain does not comprise additional disulfide bonds compared to the wild-type CH3 domain. In alternative embodiments, the modified CH3 comprises at least one disulfide bond as compared to the wild-type CH3 domain, provided that the modified CH3 has a melting temperature of 70 ℃ or greater in the absence of disulfide bonds. In one embodiment, the modified CH3 domain comprises at least one disulfide bond as compared to the wild-type CH3 domain, and the modified CH3 domain has a melting temperature (Tm) of about 77.5 ℃ or greater. In one embodiment, the modified CH3 domain comprises at least one disulfide bond as compared to the wild-type CH3 domain, and the modified CH3 domain has a melting temperature (Tm) of about 78 ℃ or greater. In another embodiment, the modified CH3 domain comprises at least one disulfide bond as compared to the wild-type CH3 domain, and the modified CH3 domain has the following melting temperature (Tm): above about 78 ℃, or above about 78.5 ℃, or above about 79 ℃, or above about 79.5 ℃, or above about 80 ℃, or above about 80.5 ℃, or above about 81 ℃, or above about 81.5 ℃, or above about 82 ℃, or above about 82.5 ℃, or above about 83 ℃.

In one embodiment, the modified CH3 domain has the following melting temperature: above about 70 ℃, or above about 70.5 ℃, or above about 71 ℃, or above about 71.5 ℃, or above about 72 ℃, or above about 72.5 ℃, or above about 73 ℃, or above about 73.5 ℃, or above about 74 ℃, or above about 74.5 ℃, or above about 75 ℃, or above about 75.5 ℃, or above about 76 ℃, or above about 76.5 ℃, or above about 77 ℃, or above about 77.5 ℃, or above about 78 ℃, or above about 78.5 ℃, or above about 79 ℃, or above about 79.5 ℃, or above about 80 ℃, or above about 80.5 ℃, or above about 81 ℃, or above about 81.5 ℃, or above about 82 ℃, or above about 82.5 ℃, or above about 83 ℃. In another embodiment, the modified CH3 domain has the following melting temperature: about 70 ℃, or about 70.5 ℃, or about 71 ℃, or about 71.5 ℃, or about 72 ℃, or about 72.5 ℃, or about 73 ℃, or about 73.5 ℃, or about 74 ℃, or about 74.5 ℃, or about 75 ℃, or about 75.5 ℃, or about 76 ℃, or about 76.5 ℃, or about 77 ℃, or about 77.5 ℃, or about 78 ℃, or about 78.5 ℃, or about 79 ℃, or about 79.5 ℃, or about 80 ℃, or about 80.5 ℃, or about 81 ℃. In yet another embodiment, the modified CH3 domain has the following melting temperature: about 70 ℃ to about 81 ℃, or about 70.5 ℃ to about 81 ℃, or about 71 ℃ to about 81 ℃, or about 71.5 ℃ to about 81 ℃, or about 72 ℃ to about 81 ℃, or about 72.5 ℃ to about 81 ℃, or about 73 ℃ to about 81 ℃, or about 73.5 ℃ to about 81 ℃, or about 74 ℃ to about 81 ℃, or about 74.5 ℃ to about 81 ℃, or about 75 ℃ to about 81 ℃, or about 75.5 ℃ to about 81 ℃, or 76 ℃ to about 81 ℃, or about 76.5 ℃ to about 81 ℃, or about 77 ℃ to about 81 ℃, or about 77.5 ℃ to about 81 ℃, or about 78 ℃ to about 81 ℃, or about 78.5 ℃ to about 82 ℃, or about 79 ℃ to about 81 ℃. In yet another embodiment, the modified CH3 domain has the following melting temperature: about 71 ℃ to about 76 ℃, or about 72 ℃ to about 76 ℃, or about 73 ℃ to about 76 ℃, or about 74 ℃ to about 76 ℃.

In addition to increased stability, the heterodimeric Fc region comprises a modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation. It is understood that these amino acid mutations that promote heterodimer formation are compared to homodimer formation. Such heterodimer formation, as compared to homodimer formation, is collectively referred to herein as "purity" or "specificity" or "heterodimer purity" or "heterodimer specificity". It is understood that heterodimer purity refers to the percentage of the desired heterodimer formed in solution under standard cell culture conditions as compared to the homodimer species formed prior to selective purification of the homodimer species. For example, a heterodimer purity of 90% indicates that 90% of the dimer species in solution is the desired heterodimer. In one embodiment, the Fc variant heterodimer has the following purity: more than about 90%, or more than about 91%, or more than about 92%, or more than about 93%, or more than about 94%, or more than about 95%, or more than about 96%, or more than about 97%, or more than about 98%, or more than about 99%. In another embodiment, the Fc variant heterodimer has the following purity: about 90%, or about 91%, or about 92%, or about 93%, or about 94%, or about 95%, or about 96%, or about 97%, or about 98%, or about 99%, or about 100%.

In a particular embodiment, an isolated heteromultimer comprising a heteromultimeric Fc region, wherein said heteromultimeric Fc region comprises a modified CH3 domain with increased stability, said modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein said modified CH3 domain has a melting temperature (Tm) of 70 ℃ or greater and the resulting heterodimer has a purity of greater than 90%. In one aspect, the obtained Fc variant heterodimer has a purity of greater than 98% and the modified CH3 domain has the following melting temperature: above about 70 ℃, or above about 71 ℃, or above about 72 ℃, or above about 73 ℃, or above about 74 ℃, or above about 75 ℃, or above about 76 ℃, or above about 77 ℃, or above about 78 ℃, or above about 79 ℃, or above about 80 ℃, or above about 81 ℃. In a further aspect, the modified CH3 domain has a melting temperature of 70 ℃ or higher and the obtained Fc variant heterodimer has the following purity: more than about 90%, or more than about 91%, or more than about 92%, or more than about 93%, or more than about 94%, or more than about 95%, or more than about 96%, or more than about 97%, or more than about 98%, or more than about 99%.

To design these Fc variants with improved stability and purity, we used an iterative process of computational design and experimental screening to select the most successful combination of positive and negative design strategies (see, figure 24).

Specifically, at the initial design stage, different negative design Fc variant heterodimers were prepared and tested for expression and stability as described in examples 1-3. The initial design phase included the Fc variant heterodimer AZ1-AZ16 (see, table 1). From this initial set of negative design Fc variant heterodimers, which are expected to have lower stability (e.g., tm of less than 71 ℃), fc variant heterodimers with greater than 90% purity and a melting temperature of about 68 ℃ or higher were selected for further development. This includes Fc variant heterodimers AZ6, AZ8 and AZ15. In the second design stage, those selected Fc variant heterodimers were further modified to drive stability and purity using positive design strategies following detailed calculations and structural analysis. The selected Fc variant heterodimers (AZ 6, AZ8, and AZ 15) were each analyzed by computational methods and comprehensive structural functional analysis to identify structural reasons for the lower stability of these Fc variants than the wild-type Fc homodimer (which was 81 ℃ for IgG 1). See table 4 for a list of Fc variant heterodimers and Tm values.

In certain embodiments, the modified CH3 domain is selected from AZ1, or AZ2, or AZ3, or AZ4, or AZ5, or AZ6, or AZ7, or AZ8, or AZ9, or AZ10, or AZ11, or AZ12, or AZ13, or AZ14, or AZ15, or AZ16. In selected embodiments, the modified CH3 domain is AZ6, or AZ8 or AZ15.

Computer tools and structure-function analysis include, but are not limited to, molecular dynamics analysis (MD), side chain/backbone repackaging, knowledge base potential (Knowledge Base Potential, KBP), cavity and (hydrophobic) packaging analysis (LJ, CCSD, SASA, dSASA (carbon/full atom)), electrostatic-GB calculation, and coupling analysis. (for an overview of computational strategies, see FIG. 24)

One aspect of the protein engineering approach relies on a combination of structural information of Fc IgG proteins derived from X-ray crystallography and computational modeling and modeling of wild-type and variant forms of the CH3 domain. This allows us to gain new structural and physicochemical insights about the potential effects of individual amino acids and their synergistic effects. These structural and physicochemical insights obtained from multiple modified CH3 domains, along with empirical data obtained regarding their stability and purity, help us develop and understand the relationship between purity and stability of Fc heterodimers compared to Fc homodimers and simulated structural models. To perform our simulations, we began by constructing a complete and realistic model and perfecting the wild-type Fc structural mass of IgG1 antibodies. Protein structures derived from X-ray crystallography lack details concerning certain characteristics of proteins in aqueous media under physiological conditions, and our perfected procedures address these limitations. These include constructing the deleted regions of the protein structure (often flexible portions of the protein such as loops and some residue side chains), evaluating and defining the proton status of neutral and charged residues and placement of potentially functionally related water molecules associated with the protein.

Molecular Dynamics (MD) algorithms are one tool we use to evaluate the intrinsic kinetic properties of Fc homodimers and modified CH3 domains in an aqueous environment by mimicking protein structure. Molecular dynamics simulation tracks the kinetic orbitals of the molecules resulting from the interactions and forces between all atomic entities in the protein and its local environment, in which case the atoms constitute Fc and its surrounding water molecules. After molecular dynamics simulation, aspects of the orbitals were analyzed to gain insight into the structural and kinetic characteristics of Fc homodimers and variant Fc heterodimers, which we used to identify specific amino acid mutations to improve the purity and stability of the molecules.

Thus, the resulting MD trajectories are studied using methods such as principal component analysis to show a pattern of motion with low natural frequencies in the Fc structure. This provides insight into the potential conformational sub-states of proteins (see, figure 32). Although the key protein-protein interaction between chains a and B in the Fc region occurs at the interface of the CH3 domains, our simulations indicate that this interface acts as a hinge in motion, which involves the "opening" and "closing" of the N-termini of the CH2 domains relative to each other. As can be seen in fig. 16, the CH2 domain interacts with FcgR at its ends. Thus, while not wishing to be bound by theory, it appears that the introduction of an amino acid mutation at the CH3 interface confers the magnitude and nature of the open/close movement at the N-terminus of Fc, and thus how Fc interacts with FcgR. See, example 4 and table 5.

The resulting MD trajectories are also studied to determine the variability of specific amino acid residue positions in Fc structures based on analyzing their flexibility profile and analyzing their environment. This algorithm enabled us to identify residues that did not affect protein structure and function, providing a unique insight into the residue characteristics and variability of subsequent design stages of the modified CH3 domain. The analysis also enables us to compare multiple simulations and evaluate variability based on outliers after analyzing the profile.

The resulting MD trajectories were also studied to determine the relevant residue movements in the protein and the formation of residue networks resulting from the coupling between them. The discovery of the kinetic correlation and residue network in Fc structures is a key step in understanding the insight that proteins act as kinetic entities and are useful for developing the effects of mutations at distant sites. See, e.g., example 6

Thus, we studied the effect of mutation on the local environment of the mutation site in detail. The formation of a well-packed core at the interface of CH3 between chains a and B is critical for spontaneous pairing of the two chains in a stable Fc structure. Good packaging is the result of strong structural complementarity between interacting chaperones together with favorable interactions between contacting groups. The favorable interactions result from the formation of buried hydrophobic contacts and/or complementary electrostatic contacts between hydrophilic polar groups that well remove solvent exposure. These hydrophobic and hydrophilic contacts have an entropy and enthalpy contribution to the free energy of dimer formation at the CH3 interface. We have used various algorithms to accurately model the packaging at the CH3 interface between chains a and B and then evaluate the thermodynamic properties of the interface by scoring some relevant physicochemical properties.

We have used some protein packaging methods, including flexible backbones, to optimize and prepare model structures for a number of variants for our computational screening. After packaging, we evaluated a number of items including contact density, collision score, hydrogen bonding, hydrophobicity, and static electricity. The use of a solvation model enables us to more accurately address the effects of solvent environment and contrast free energy differences after mutation of specific positions in the protein to alternative residue types. The contact density and collision score provide a measure of complementarity (a key aspect of effective protein packaging). These screening programs are based on knowledge-based potential or application of coupling analysis schemes, which rely on pairwise residue interaction energies and entropy calculations.

This comprehensive on-chip computer analysis provides a more detailed understanding of the differences in each Fc variant compared to wild type with respect to ligation hotspots, asymmetric sites, cavities and poorly packaged regions, structural dynamics of individual sites, and local unfolding sites. These combined results of the computational analysis identified specific residues, sequence/structural motifs and cavities that were not optimized and combined for lower specificity responsible for lower stability (e.g., tm at 68 ℃) and/or <90% purity. In the second design phase we used a targeted positive design to specifically address these assumptions by additional point mutations, and tested these by on-chip engineering using the methods and assays described above (see fig. 24). As described in examples 1-4, experiments demonstrated that the expression and stability of the Fc variant heterodimer (Fc variant heterodimer AZ17-AZ 101) was designed in the second stage for each targeting design to improve stability and purity.

In certain embodiments, provided herein are isolated heteromultimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain having increased stability, the modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain is AZ17, or AZ18, or AZ19, or AZ20, or AZ21, or AZ22, or AZ23, or AZ24, or AZ25, or AZ26, or AZ27, or AZ28, or AZ29, or AZ30, or AZ21, or AZ32, or AZ33, or AZ34, or AZ35, or AZ36, or AZ37, or AZ38, or AZ39, or AZ40, or AZ41, or AZ42, or AZ43, or AZ44, or AZ45, or AZ46, or AZ47, or AZ48, or AZ49, or AZ50, or AZ51, or AZ52, or AZ53, or AZ54, or AZ55, or AZ56, or AZ 57; or AZ58, or AZ59, or AZ60, or AZ61, or AZ62, or AZ63, or AZ64, or AZ65, or AZ66, or AZ67, or AZ68, or AZ69, or AZ70, or AZ71, or AZ72, or AZ73, or AZ74, or AZ75, or AZ76, or AZ77, or AZ78, or AZ79, or AZ80, or AZ81, or AZ82, or AZ83, or AZ84, or AZ85, or AZ86, or AZ87, or AZ88, or AZ89, or AZ90, or AZ91, or AZ92, or AZ93, or AZ94, or AZ95, or AZ96, or AZ97, or AZ98, or AZ99, or AZ100, or AZ101. In one exemplary embodiment of the present invention, the modified CH3 domain is AZ17, or AZ18, or AZ19, or AZ20, or AZ21, or AZ22, or AZ23, or AZ24, or AZ25, or AZ26, or AZ27, or AZ28, or AZ29, or AZ30, or AZ21, or AZ32, or AZ33, or AZ34, or AZ38, or AZ42, or AZ43, or AZ44, or AZ45, or AZ46, or AZ47, or AZ48, or AZ49, or AZ50, or AZ52, or AZ53, or AZ54, or AZ58, or AZ59, or AZ60, or AZ 61; or AZ62, or AZ63, or AZ64, or AZ65, or AZ66, or AZ67, or AZ68, or AZ69, or AZ70, or AZ71, or AZ72, or AZ73, or AZ74, or AZ75, or AZ76, or AZ77, or AZ78, or AZ79, or AZ81, or AZ82, or AZ83, or AZ84, or AZ85, or AZ86, or AZ87, or AZ88, or AZ89, or AZ91, or AZ92, or AZ93, or AZ94, or AZ95, or AZ98, or AZ99, or AZ100, or AZ101. In a particular embodiment, the modified CH3 domain is AZ33 or AZ34. In another embodiment, the modified CH3 domain is AZ70 or AZ90.

In one exemplary embodiment, the CH3 domain comprises first and second polypeptides (also referred to herein as chain a and chain B), wherein the first polypeptide comprises amino acid modifications L351Y, F a and Y407V, and wherein the second polypeptide comprises amino acid modifications T366I, K392M and T394W. In another embodiment, the first polypeptide comprises amino acid modifications L351Y, S400E, F a and Y407V and the second polypeptide comprises amino acid modifications T366I, N390R, K392M and T394W.

This iterative process of computational structure-function analysis, targeted engineering and experimental validation was used in the subsequent design stage to design the remaining Fc variants listed in table 1 and resulted in Fc variant heterodimers with greater than 90% purity and increased stability with CH3 domain melting temperatures greater than 70 ℃. In certain embodiments, the Fc variant comprises an amino acid mutation selected from AZ1 to AZ 136. In further embodiments, the Fc variant comprises an amino acid mutation selected from the Fc variants listed in table 4.

Two core scaffolds, scaffold 1 and scaffold 2, were identified from the first and second design stages, with additional amino acid modifications introduced into these scaffolds to fine tune the purity and stability of the Fc variant heterodimer. For a detailed description of development of scaffold 1 including AZ8, AZ17-62 and the variants listed in table 6, see example 5. For a detailed description of development of scaffold 2 including AZ15 and AZ63-101 and the variants listed in table 7, see example 6.

The core mutation of stent 1 comprises L351Y_F405A_Y407V/T394W. Scaffold 1a comprises amino acid mutation T366i_k392m_t394W/f405a_y407V and scaffold 1b comprises amino acid mutation T366l_k392m_t394W/f405a_y407V. See, example 5.

In certain embodiments, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second polypeptide comprises amino acid modification T394W. In one aspect, the modified CH3 domain further comprises a point mutation at position F405 and/or K392. These mutations at position K392 include, but are not limited to, K392V, K392M, K392R, K392L, K392F or K392E. These mutations at position F405 include, but are not limited to, F405I, F405M, F405S, F405S, F V or F405W. In another aspect, the modified CH3 domain further comprises a point mutation at position T411 and/or S400. These mutations at position T411 include, but are not limited to, T411N, T411R, T411Q, T411K, T D, T E or T411W. These mutations at position S400 include, but are not limited to, S400E, S400D, S R or S400K. In yet another embodiment, the modified CH3 domain comprises a first and a second polypeptide, wherein the first polypeptide comprises amino acid modifications L351Y, F a and Y407V and the second polypeptide comprises amino acid modification T394W, wherein the first and/or second polypeptide comprises a further amino acid modification at position T366 and/or L368. These mutations at position T366 include, but are not limited to, T366A, T366I, T366L, T366M, T366Y, T366S, T366C, T V or T366W. In one exemplary embodiment, the amino acid mutation at position T366 is T366I. In another exemplary embodiment, the amino acid mutation at position T366 is T366L. The mutations at position L368 are L368D, L368R, L368T, L M, L368V, L368F, L S and L368A.

In certain embodiments, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second polypeptide comprises amino acid modifications T366L and T394W. In another embodiment, the modified CH3 domain comprises a first and a second polypeptide, wherein the first polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second polypeptide comprises amino acid modifications T366I and T394W.

In certain other embodiments, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second polypeptide comprises amino acid modifications T366L, K392M and T394W. In another embodiment, the modified CH3 domain comprises a first and a second polypeptide, wherein the first polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second polypeptide comprises amino acid modifications T366I, K392M and T394W.

In yet another embodiment, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modifications F405A and Y407V and the second polypeptide comprises amino acid modifications T366L, K392M and T394W. In another embodiment, the modified CH3 domain comprises a first and a second polypeptide, wherein the first polypeptide comprises amino acid modifications F405A and Y407V and the second polypeptide comprises amino acid modifications T366I, K392M and T394W.

In certain embodiments, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modifications F405A and Y407V, and the second polypeptide comprises amino acid modifications T366L and T394W. In another embodiment, the modified CH3 domain comprises a first and a second polypeptide, wherein the first polypeptide comprises amino acid modifications F405A and Y407V and the second polypeptide comprises amino acid modifications T366I and T394W.

In exemplary embodiments, provided herein are isolated heterodimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain with increased stability comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) of about 74 ℃ or greater. In another embodiment, provided herein is an isolated heterodimer comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain having increased stability comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) of about 74 ℃ or greater and the heterodimer has a purity of about 98% or greater.

In certain embodiments, an isolated heterodimer comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain with increased stability, the modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) above 70 ℃ and the modified CH3 domain is selected from table 6.

The core mutation of scaffold 2 comprises L351Y_Y407A/T366A_K409F. Scaffold 2a comprises the amino acid mutation L351Y_Y407A/T366V_K409F and scaffold 2b comprises the amino acid mutation Y407A/T366A_K409F. See example 6.

In certain embodiments, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modifications L351Y and Y407A and the second polypeptide comprises amino acid modifications T366A and K409F. In one aspect, the modified CH3 domain further comprises point mutations at positions T366, L351 and Y407. These mutations at position T366 include, but are not limited to, T366I, T366L, T366M, T366Y, T366S, T366C, T V or T366W. In a particular embodiment, the mutation at position T366 is T366V. Mutations at position L351 include, but are not limited to, L351I, L351D, L351R or L351F. Mutations at position Y407 include, but are not limited to, Y407V or Y407S. See, tables 1 and 4 and example 6 for the CH3 variant AZ63-AZ70.

In exemplary embodiments, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modifications L351Y and Y407A and the second polypeptide comprises amino acid modifications T366V and K409F.

In exemplary embodiments, provided herein are isolated heterodimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain with increased stability comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) of about 75.5 ℃ or greater. In another embodiment, provided herein is an isolated heterodimer comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain with increased stability comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) of about 75 ℃ or greater and the heterodimer has a purity of about 90% or greater.

In certain other embodiments, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modifications L351Y and Y407A and the second polypeptide comprises amino acid modifications T366A and K409F, wherein the modified CH3 domain comprises one or more amino acid modifications at positions T411, D399, S400, F405, N390, and/or K392. These mutations at position D399 include, but are not limited to, D399R, D399W, D399Y or D399K. Mutations at position T411 include, but are not limited to, T411N, T411R, T411Q, T411K, T411D, T E or T411W. Mutations at position S400 include, but are not limited to, S400E, S400D, S400R or S400K. Mutations at position F405 include, but are not limited to, F405I, F405M, F405S, F405S, F V or F405W. Mutations at position N390 include, but are not limited to, N390R, N390K or N390D. Mutations at position K392 include, but are not limited to, K392V, K392M, K392R, K392L, K392F or K392E. See, tables 1 and 4 and CH3 variants AZ71-101 in example 6.

In exemplary embodiments, the modified CH3 domain comprises first and second polypeptides (also referred to herein as chains a and B), wherein the first polypeptide comprises amino acid modification Y407A and the second polypeptide comprises amino acid modifications T366A and K409F. In one aspect, this modified CH3 domain further comprises the amino acid modifications K392E, T411E, D399R and S400R. In another embodiment, the modified CH3 domain comprises a first and a second polypeptide, wherein the first polypeptide comprises amino acid modifications D399R, S R and Y407A and the second polypeptide comprises amino acid modifications T366A, K409F, K392E and T411E.

In exemplary embodiments, provided herein are isolated heterodimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain with increased stability comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) of about 74 ℃ or greater. In another embodiment, provided herein is an isolated heterodimer comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain with increased stability comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) of about 74 ℃ or greater and the heterodimer has a purity of about 95% or greater.

In certain embodiments, provided herein are isolated heterodimers comprising a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain with increased stability comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) above 70 ℃ and the modified CH3 domain is selected from table 7.

Furthermore, this new approach to designing Fc variant heterodimers with improved stability and purity can be applied to other types and isoforms of Fc regions. In certain embodiments, the Fc region is a human IgG Fc region. In further embodiments, the human IgG Fc region is a human IgGI, igG2, igG3, or IgG4Fc region. In some embodiments, the Fc region is from an immunoglobulin selected from IgG, igA, igD, igE and IgM. In some embodiments, the IgG is a subtype selected from the group consisting of IgG1, igG2a, igG2b, igG3, and IgG 4.

Table 1.1: CH3 domain amino acid modification for Fc variant heterodimer formation

/>

Table 1.2: CH3 domain amino acid modifications for use in generating Fc variant heterodimers. The DSC melting temperature of the CH3 domain was evaluated as shown in FIGS. 29A-29B and described in the examples.

Table 1.3: CH3 domain amino acid modifications for use in generating Fc variant heterodimers. Kd in the above table was determined as described in the examples and in fig. 35.

The Fc region described herein comprises a CH3 domain or fragment thereof, and may additionally comprise one or more additional constant region domains or fragments thereof, including a hinge, CH1, or CH2. It will be appreciated that numbering of Fc amino acid residues is that of the EU index as in Kabat et al 1991,NIH Publication 91-3242,National Technical Information Service,Springfield,Va. The EU index as described in "Kabat" refers to the EU index numbering of human IgG1 Kabat antibodies. For convenience, table B provides amino acids from the CH2 and CH3 domains of human IgG1, numbered according to the EU index as set forth in Kabat.

Table B

According to one aspect of the invention there is provided an isolated heterodimeric Fc construct comprising a modified heterodimeric CH3 domain, the modified CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides comprises an amino acid modification of K392J, wherein J is selected from L, I or an amino acid having a side chain volume that is not substantially greater than K; wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 74 ≡c and a purity of at least 95%; and wherein at least one amino acid modification is not a modification of an amino acid at the interface between the first and the second CH3 domain polypeptides. In certain embodiments are heterodimeric Fc constructs described herein comprising at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments are isolated heterodimeric Fc constructs described herein comprising at least one T350V modification. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a Tm of about 77 ℃ or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80 ℃ or greater. In certain embodiments, there is provided an isolated heterodimeric Fc construct described herein, wherein the at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of L351, F405, and Y407. In some embodiments are isolated heterodimeric Fc constructs wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide further comprising an amino acid modification of T366. In certain embodiments are isolated heterodimeric Fc constructs described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions L351, F405, and Y407, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions T366, K392, and T394. In embodiments are isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second CH3 domain polypeptide comprises amino acid modifications T366L, K392M and T394W. In some embodiments are isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second CH3 domain polypeptide comprises amino acid modifications T366L, K392L and T394W. In another embodiment is an isolated heterodimeric Fc construct described herein, the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second CH3 domain polypeptide comprises amino acid modifications T366I, K392M and T394W. In some embodiments are isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V, and the second CH3 domain polypeptide comprises amino acid modifications T366I, K392L and T394W. In certain embodiments are isolated heterodimeric Fc constructs described herein, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position S400. In another embodiment is an isolated heterodimeric Fc construct described herein comprising modification S400Z, wherein Z is selected from the group consisting of a positively charged amino acid and a negatively charged amino acid. In some embodiments, the positively charged amino acid is lysine or arginine and the negatively charged amino acid is aspartic acid or glutamic acid. In certain embodiments are isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide comprising an amino acid modification selected from S400E and S400R. In some embodiments, there is provided an isolated heterodimeric Fc construct described herein, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position N390. In some embodiments, the modification of N390 is N390Z, wherein Z is selected from the group consisting of a positively charged amino acid and a negatively charged amino acid. In embodiments, N390Z is N390R. In certain embodiments of the isolated heterodimeric Fc constructs described herein, the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification S400E and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification N390R. In some embodiments of the isolated heterodimeric Fc constructs described herein, each of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide, one of which comprises the amino acid modification Q347R and the other modified CH3 domain polypeptide comprises the amino acid modification K360E.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified heterodimeric CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides comprises an amino acid modification of K392J, wherein J is selected from L, I or an amino acid having a side chain volume that is not substantially greater than K; wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 74 ≡c and a purity of at least 95%; and wherein at least one amino acid modification is not a modification of an amino acid at the interface between the first and the second CH3 domain polypeptides. In certain embodiments are heterodimeric Fc constructs described herein comprising at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments are isolated heterodimeric Fc constructs described herein comprising at least one T350V modification. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a Tm of about 77 ℃ or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80 ℃ or greater. In embodiments are isolated heterodimeric Fc constructs described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of K409 and T411. In certain embodiments is an isolated heterodimeric Fc construct described herein comprising at least one of K409F, T E and T411D. In some embodiments are isolated heterodimeric Fc constructs described herein, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of D399. In some embodiments, the amino acid modification of D399 is at least one of D399R and D399K.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified heterodimeric CH3 domain comprising: a first modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide; wherein at least one of the first and second CH3 domain polypeptides comprises an amino acid modification of K392J, wherein J is selected from L, I or an amino acid having a side chain volume that is not substantially greater than K; wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 74 ℃ and a purity of at least 95%; and wherein at least one amino acid modification is not a modification of an amino acid at the interface between the first and the second CH3 domain polypeptides. In certain embodiments are heterodimeric Fc constructs described herein comprising at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof. In some embodiments are isolated heterodimeric Fc constructs described herein comprising at least one T350V modification. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater. In embodiments are isolated heterodimeric Fc constructs described herein, wherein the modified CH3 domain has a Tm of about 77 ℃ or greater. In certain embodiments, the modified CH3 domain has a Tm of about 80 ℃ or greater. In certain embodiments of the isolated heterodimeric Fc constructs described herein, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of K409F, T E and T411D, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of Y407A, Y407I, Y V, D399R and D399K. In some embodiments is any of the isolated heterodimeric Fc constructs described herein further comprising a first modified CH3 domain comprising one of the amino acid modifications T366V, T366I, T366A, T M and T366L; and a second modified CH3 domain comprising the amino acid modification L351Y. In some embodiments is any of the isolated heterodimeric Fc constructs described herein comprising a first modified CH3 domain comprising one of the amino acid modifications K392L or K392E; and a second modified CH3 domain comprising one of the amino acid modifications S400R or S400V.

Provided herein are isolated heterodimeric Fc constructs comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least four amino acid mutations, wherein at least one of the first and the second modified CH3 domain polypeptides comprises a mutation selected from N390Z and S400Z, wherein Z is selected from a positively charged amino acid and a negatively charged amino acid, and wherein the first and the second modified CH3 domain polypeptides preferentially form a heterodimeric CH3 domain having a melting temperature (Tm) of at least about 70 +.c and a purity of at least 90%. In embodiments, an isolated heterodimeric Fc construct is provided wherein the first modified CH3 domain polypeptide comprises an amino acid modification at positions F405 and Y407 and the second modified CH3 domain polypeptide comprises an amino acid modification at position T394. In embodiments, isolated heterodimeric Fc constructs are provided, wherein the first modified CH3 domain polypeptide comprises an amino acid modification at position L351. In certain embodiments are isolated heterodimers described herein, the second modified CH3 domain polypeptide comprising a modification at least one of positions T366 and K392. In some embodiments are isolated heterodimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ≡c and is formed at a purity of at least about 95%. In certain embodiments are isolated heterodimers described herein, and the at least one modified CH3 domain polypeptide comprises an amino acid modification of at least one of N390R, S E and S400R. In some embodiments are isolated heterodimers described herein, one of the first and second modified CH3 domain polypeptides comprising an amino acid modification at position 347 and the other modified CH3 domain polypeptide comprising an amino acid modification at position 360. In certain embodiments are isolated heterodimers described herein, at least one of the first and second modified CH3 domain polypeptides comprising an amino acid modification of T350V. In a particular embodiment is an isolated heterodimer as described herein, the first modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of L351Y, F a and Y407V; and the second modified CH3 domain polypeptide comprises at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In certain embodiments described herein are isolated heterodimers, the first modified CH3 domain polypeptide comprises amino acid modifications at positions D399 and Y407, and the second modified CH3 domain polypeptide comprises amino acid modifications at positions K409 and T411. In some embodiments are isolated heterodimers described herein, a first CH3 domain polypeptide comprises an amino acid modification at position L351, and a second modified CH3 domain polypeptide comprises amino acid modifications at positions T366 and K392. In a particular embodiment are isolated heterodimers as described herein, at least one of the first and second CH3 domain polypeptides comprising an amino acid modification of T350V. In certain embodiments are isolated heterodimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 750C or greater and is formed at a purity of at least about 95%. In certain embodiments, provided are isolated heterodimeric Fc constructs described herein, the first modified CH3 domain polypeptide comprising an amino acid modification selected from the group consisting of L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A and Y407V; and the second modified CH3 domain polypeptide comprises an amino acid modification selected from the group consisting of T366V, T366I, T366L, T366M, N390D, N390 42392E, K L, K392I, K D, K E, K409F, K409W, T D and T411E.

Provided herein are isolated heterodimeric Fc constructs comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least three amino acid mutations, wherein one of the first and the second modified CH3 domain polypeptides comprises a mutation selected from T411E and T411D, and wherein the first and the second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 70 ≡c and a purity of at least 90%. In embodiments, an isolated heterodimeric Fc construct is provided wherein the first modified CH3 domain polypeptide comprises an amino acid modification at positions F405 and Y407 and the second modified CH3 domain polypeptide comprises an amino acid modification at position T394. In embodiments, isolated heterodimeric Fc constructs are provided, with the first modified CH3 domain polypeptide comprising an amino acid modification at position L351. In certain embodiments are isolated heterodimers described herein, the second modified CH3 domain polypeptide comprising a modification at least one of positions T366 and K392. In some embodiments are isolated heterodimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ≡c and is formed at a purity of at least about 95%. In certain embodiments are isolated heterodimers described herein, and the at least one modified CH3 domain polypeptide comprises an amino acid modification of at least one of N390R, S E and S400R. In some embodiments are isolated heterodimers described herein, one of the first and second modified CH3 domain polypeptides comprising an amino acid modification at position 347 and the other modified CH3 domain polypeptide comprising an amino acid modification at position 360. In certain embodiments are isolated heterodimers described herein, at least one of the first and second modified CH3 domain polypeptides comprising an amino acid modification of T350V. In a particular embodiment is an isolated heterodimer as described herein, the first modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of L351Y, F a and Y407V; the second modified CH3 domain polypeptide comprises at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W. In certain embodiments described herein are isolated heterodimers, a first modified CH3 domain polypeptide comprising amino acid modifications at positions D399 and Y407, and a second modified CH3 domain polypeptide comprising amino acid modifications at positions K409 and T411. In some embodiments are isolated heterodimers described herein, a first CH3 domain polypeptide comprises an amino acid modification at position L351, and a second modified CH3 domain polypeptide comprises amino acid modifications at positions T366 and K392. In a particular embodiment are isolated heterodimers as described herein, at least one of the first and second CH3 domain polypeptides comprising an amino acid modification of T350V. In certain embodiments are isolated heterodimers described herein, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater and is formed at a purity of at least about 95%. In certain embodiments, provided are isolated heterodimeric Fc constructs described herein, the first modified CH3 domain polypeptide comprising an amino acid modification selected from the group consisting of L351Y, D399R, D399K, S400D, S400E, S400R, S400K, Y407A and Y407V; and the second modified CH3 domain polypeptide comprises an amino acid modification selected from the group consisting of T366V, T366I, T366L, T366M, N390D, N390 42392E, K L, K392I, K D, K E, K409F, K409W, T D and T411E.

Provided herein are isolated heterodimeric Fc constructs comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366I, K392M and T394W.

In one aspect, there is provided an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide of amino acid modifications T366I, K L and T394W.

In certain embodiments, the Fc variant comprises a CH2 domain. In some embodiments, the CH2 domain is a variant CH2 domain. In some embodiments, the variant CH2 domain comprises asymmetric amino acid substitutions in the first and/or second polypeptide chains. In some embodiments, the heteromultimer comprises asymmetric amino acid substitutions in the CH2 domain such that one chain of the heteromultimer selectively binds to an Fc receptor.

In certain embodiments, the heteromultimer selectively binds to an Fc receptor. In some embodiments, the Fc receptor is a member of the fcγ receptor family. In some embodiments, the receptor is selected from fcyri, fcyriia, fcyriib, fcyriic, fcyriiia, and fcyriiib. In one embodiment, the CH2 domain comprises asymmetric amino acid modifications that promote selective binding to fcγ receptors.

In some embodiments, the heteromultimer binds selectively to fcγriiia. In some embodiments, the heteromultimer comprises an asymmetric amino acid substitution selected from S267D, K392D and K409D. In some embodiments, the heteromultimer binds selectively to fcyriia. In some embodiments, the heteromultimer comprises an asymmetric amino acid substitution selected from the group consisting of S239D, K326E, A L and I332E. In some embodiments, the heteromultimer binds selectively to fcyriib. In some embodiments, the heteromultimer comprises an asymmetric amino acid substitution selected from the group consisting of S239D, D265S, E269K and I332E. In some embodiments, the heteromultimer binds selectively to fcγriiia and fcγriia. In some embodiments, the heteromultimer comprises an asymmetric amino acid substitution selected from the group consisting of S239D, D S and S298A. In some embodiments, the heteromultimer binds selectively to fcγriiia and fcγriib. In some embodiments, the heteromultimer comprises an asymmetric amino acid substitution selected from the group consisting of S239D, S298A, K326E, A L and I332E. In some embodiments, the heteromultimer binds selectively to fcyriia and fcyriib. In some embodiments, the heteromultimer comprises an asymmetric amino acid substitution selected from the group consisting of S239D, D265S, S298A and I332E.

In certain embodiments, methods of designing multifunctional therapeutics comprising the heteromultimers described herein are provided. In some embodiments, methods of designing bifunctional therapeutic agents comprising variant Fc heterodimers are provided. In some embodiments, methods are provided for designing asymmetric mutations in the CH3 domain of variant Fc heterodimers derived with mutations in the CH2 domain. In some embodiments, methods of designing selectivity for different fcγ receptors based on mutations in asymmetric fcs are provided. In certain embodiments, methods of designing mutations that favor binding of fcγ receptors to one side of an Fc molecule are provided. In certain embodiments, methods of designing polar drivers that favor interactions of fcγ receptors with only one side of an asymmetric Fc scaffold of a heteromultimer described herein are provided.

In some embodiments, polypeptides are provided that comprise mutations in the CH2 domain of an asymmetric Fc that result in a preferential fcγ receptor selectivity profile. In some embodiments, the mutation in the CH3 domain results in preferential formation of heterodimeric Fc. In certain embodiments are methods of designing bispecific therapeutic entities based on asymmetric fcs described herein. In certain embodiments are methods of designing a multispecific therapeutic entity based on an asymmetric Fc described herein.

Monoclonal antibodies, such as IgG, are symmetrical molecules composed of two identical heavy polypeptide chains and two light polypeptide chains (fig. 14), each comprising multiple immunoglobulin (Ig) domains. The IgG class of mabs exists in one of four isotypes (IgG 1, igG2, igG3, or IgG 4). The heavy chain consists of four Ig domains (VH, CH1, CH2 and CH 3), and the light chain consists of two (VL and CL) Ig domains, respectively. The VH and CH1 domains from each heavy chain combine with the VL and CL domains of the light chain to form the two Fab ("fragment antigen binding") arms of the mAb. The CH3 and CH2 domains of the two heavy chains interact via protein-protein contact across the CH3 domain and glycosylation in the CH2 domain to form a homodimerized Fc ("crystallizable fragment") region. The linker region between the CH1 and CH2 domains of an antibody constitutes the hinge region of an antibody molecule. In addition to linking the Fab and Fc regions of the mAb, the hinge also maintains disulfide linkages across the two heavy chains and holds them together. The number of amino acids and disulfide linkages in the hinge region varies significantly between the four isotypes of IgG. The glycosylation pattern in IgG molecules can be significantly different, with about 30 different carbohydrate moieties having been observed in IgG molecules [ Arnold j.n.; wormald m.r.; sim r.b.; rudd P.M. and Dwek R.A. (2007) Annual Reviews of Immunology 25,21-50].

The symmetrical nature of the monoclonal antibody structure results in both Fab arms having antigen binding capacity affinity that matures to recognize the same epitope. At the other end, the Fc portion of the antibody molecule is involved in interactions with various receptor molecules on immune or "effector" cells, and some of these interactions are responsible for mediating effector functions such as antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), and complement activation. In general, effector functions involve immune responses that lead to pathogen or toxin neutralization and elimination, complement activation, and phagocytic responses from the humoral immune system. Fcγ receptor (fcγr) molecules on effector cells contact the Fc of IgG antibodies involved in activation of the intact antigen-antibody immune complex to mediate and modulate the effector response. Optimizing the interaction of monoclonal antibody-based protein therapeutics with these fcγ receptors can lead to improved efficacy of these drug candidates.

In humans, there are three known classes of fcγr, each with a further polymorphic type within it. Fc in IgG1 molecules is known to bind fcγri (CD 64) with dissociation constants in the nanomolar range, whereas fcγrii (CD 32) and fcγriii (CD 16) binding occurs in the micromolar range [ Bruhns p.; iannascoli b; england P.; mancardi d.a.; fernandez n; jorieux s and Daeron m (2009) Blood 113:3716-25]. The high affinity fcyri receptor can bind IgG in monomeric form, while the low affinity fcyrii and fcyriii receptors can only bind antigen-antibody immune complexes or IgG aggregates due to the avidity effect of the antibodies. Different IgG forms have different affinities for different fcγrs; specifically, igG1 and IgG3 exhibit stronger activity. Fcγ receptors are extracellular domains of transmembrane proteins and have cytoplasmic domains involved in regulating intracellular signaling pathways. When bound to antibody-mediated immune complexes, accumulate on the surface of immune cells, these molecules modulate the effector response according to the nature of the signaling unit linked to fcγr on the cytoplasmic end of these cell surface receptors [ Nimmerjahn f. And Ravetch j.v. (2008) Nature Immu Rev (1): 34-47].

At the human chromosome level, three genes encode fcγri (fcγria, fcγrib, fcγric) and fcγrii (fcγriia, fcγriib, fcγriic) and two genes encode fcγriii (fcγriiia, fcγriiib). In human fcγ receptors that bind IgG, fcγria, fcγric and fcγriiia types have been shown to be associated with a common γ -chain signal adaptor protein membrane, which contains an activation motif (ITAM) based on cytoplasmic immune receptor tyrosine, which leads to activation of effector functions. Fcγriia and fcγriic also comprise cytoplasmic ITAMs, but no common γ -chain signal adapter protein. Meanwhile, fcyriib is linked to an immunoreceptor tyrosine based inhibitory motif (ITIM). Activation of fcγriib, which results in ITIM phosphorylation, results in inhibition of the activation signaling cascade. Fcγriiib, despite the lack of tyrosine-based immunomodulatory cytoplasmic tails, has a GPI (glycosyl-phosphatidyl-inositol) anchor and has been shown to assist in activating some granulocytes in the presence of fcγriia.

Table C: fcγ receptor characterization

ITAM: an immunoreceptor tyrosine-based activation motif; ITIM: an immunoreceptor tyrosine-based inhibitory motif; GPI glycosyl phosphatidylinositol

Although the functional roles of ITAM and ITIM motifs and related receptor molecules are known, the nature and mechanism of regulation of combined signaling is not fully understood, particularly when combined with the activity of many other immune cell surface receptors and adapter molecules (e.g., BCR, CD22, CD45, etc.) involved in signal transduction. Against this background, designing Fc-like molecules that can interact with these fcγ receptors with a fine selectivity profile is a valuable scaffold in any attempt to deconvolute and modulate the effects of such receptor molecules with fine-tuning activity.

In the context of designing antibody molecules that can distinguish fcγr, the following facts complicate efforts: extracellular Fc binding portions of fcyrii and fcyriii receptor types exhibit a high degree of sequence similarity (fig. 15), which may be attributed at least in part to ancestral segment-wise repeats. The two major types of fcγrii receptors, a and B, have 69% sequence identity, whereas fcγriia and fcγriiia exhibit about 44% sequence identity. Fcγriib and fcγriic differ only in 2 residues of the extracellular region, although their intracellular regions differ significantly in the presence of ITIM and ITAM motifs, respectively. Thus, it is expected that therapeutic antibody molecules that need to bind to one receptor may also bind to other receptor types, possibly resulting in an undesired therapeutic effect.

Further complicating the problem, each receptor type presents multiple Single Nucleotide Polymorphisms (SNPs) and Copy Number Variations (CNVs). The diversity of receptors obtained affects differentially its affinity for IgG and its mechanism of action. These genetic changes can affect the affinity of a particular IgG subclass for fcγ receptors, alter downstream effector events, or affect mechanisms that alter receptor expression levels, resulting in functionally related phenotypically, nonfunctional, or functionally unknown receptor variants (Bournazos s.; woof j.m.; hart s.p. and DRANSFIELD i. (2009) CLINICAL AND Experimental Immunology (2): 244-54). They may lead to complex effects, altering the balance between activation and inhibition of receptor signaling, leading to the generation of disease-susceptible phenotypes.

Some of these allelic variations are listed in table C. Notably, the R131 variant in fcyriia is a high responder with IgG1, while alternative H131 variants show more efficient interactions with IgG2 and IgG 3. In the case of fcγriiia, increased NK cell activity was demonstrated for the V-homozygous donor at position 158 compared to the homozygous F/F158 individual, due to the higher affinity of the former allotype for human IgG1, igG3 and IgG 4. Allelic variants NA1 and NA2 of FcgammaRIIIb are the result of tetramino acid substitutions which in turn lead to differences in receptor glycosylation. NA1 alleles exhibit enhanced binding and phagocytosis by the immune complex of neutrophils. Fcyriib has two known allelic variants 232I and 232T. The negative regulatory activity of the 232T variant is known to be strongly impaired. The frequency of fcγr polymorphisms and their association with differential responses to susceptibility to infection or disease conditions such as Systemic Lupus Erythematosus (SLE), rheumatoid Arthritis (RA), vasculitis, immune-mediated thrombocytopenic purpura (ITP), myasthenia gravis, multiple Sclerosis (MS), and immune neuropathy (gill-barre syndrome (GBS)) have been reported.

Copy number changes in fcγr genes, particularly for fcγriiib, fcγriic and fcγriiia have been shown, and further correlation of these differences with cell surface expression of these receptors has been noted. In contrast, fcγriia and fcγriib show no change in gene copy number. Indeed, low copy numbers of fcγriiib have been associated with glomerulonephritis in the autoimmune disease Systemic Lupus Erythematosus (SLE) [ Aitman TJ et al (2006) Nature 16;439 (7078):851-5]. This is particularly interesting in view of the fact that the non-signaling GPI module anchors the fcγriiib receptor. It is speculated that the presence of these fcyriiib receptors can potentially act as competitive inhibitors of Fc interactions with other signaling fcγrs. The effect of copy number variation in fcγriic is also particularly interesting. The C/T SNP at position 202 in FcgammaRIIC converts the glutamine residue to a stop codon, preventing the production of a functional protein. The functional open reading frame of fcγriic is expressed in 9% of healthy individuals (white population) and there is a significantly excessive proportion of this gene present in the ITP population (19%), suggesting a propensity for these phenotypes to ITP [ Breunis WB et al (2008) Blood111 (3): 1029-38]. It has been shown that these receptors mediate achieved ADCC to a greater extent than fcyriiia in individuals expressing functional fcyriic on NK cells. Such complexities associated with these polymorphisms and genetic changes highlight the need for personalized therapeutic strategies, requiring highly tailored therapeutic agents.

Various effector cells differ in these fcγ receptors and in their humoral and tissue distribution, thus contributing to their activation and changes in the mechanism of action [ table D ]. Tuning the effect of therapeutic antibodies on the recognition of the selectivity of a particular fcγr type and the modulation of certain classes of effector cells results in optimization of the effector mechanisms for a particular disease condition. Depending on the disease condition being treated, this is intended to selectively activate or inhibit a particular effector form.

Table D: cell distribution of fcγr

In addition, fcγr is also expressed by follicular dendritic cells, endothelial cells, microglia, osteoclasts and mesangial cells. Currently, the functional significance of fcγr expression on these other cells is unknown.

High affinity fcyri consists of three C-type immunoglobulin superfamily (IgSF) domains, while low affinity fcyrii and fcyriii consist of two C-type IgSF domains each. The structures of fcyriia, fcyriib, fcyriiia and fcyriiib receptor proteins have been resolved by crystallography. The two IgSF domains in these structures are located 50-55 degrees relative to each other and are connected by a hinge.

The publicly available structure of Fc-fcγr co-complexes is that of the Fc-fcγriiib system, and fcγr geometry in the complex remains very close to that observed in the apo state of the protein [ Sondermann p.; huber r; oosthuizen v. and Jacob u. (2000) Nature 406, 267-273; radaev s; motyaka s; fridman w; sautes-Fridman c.and Sun p.d. (2001) J Biol Chem 276,16469-16477; sondermann P. et al Biochem Soc Trans.2002 Aug;30(4):481-6;Sondermann P,Oosthuizen V.Immunol Lett.2002 Jun 3;82(1-2):51-6; Radaev S,Sun P.Mol Immunol.2002 May;38(14):1073-83.][ FIG. 16]. The strong sequence and structural similarity between receptors forms the basis of a comparative model of Fc binding to other receptors. On the other hand, the sequence and structural similarity between these receptor molecules also makes it challenging to design fcs with fine selectivity between the receptor and its different isoforms.

Prior to evaluating the structure of Fc-fcγr complexes based on crystallography, the following problems exist: whether the 2-fold symmetry axis in an Fc molecule implies two potential binding sites for Fc-fcγr binding and an effective 2:1 stoichiometric ratio. Structural studies of Fc-FcgammaR interactions based on Nuclear Magnetic Resonance (NMR) have shown that binding of Fc to one FcgammaR on one face of the molecule induces conformational changes that preclude binding of a second FcgammaR molecule to the Fc of the same antibody molecule [ Kato K. Et al (2000) J Mol biol.295 (2): 213-24]. The geometry of the available co-crystal complex of Fc-FcgammaRIIIb verifies the binding of FcgammaR to Fc in a 1:1 stoichiometric asymmetric orientation. As shown in fig. 16, fcγr binds to the cleft on one end of the horseshoe-shaped Fc molecule and contacts the CH2 domains from both chains.

Alanine scanning mutagenesis [ SHIELDS RL et al (2001) JBC 276 (9): 6591-604] provides insight regarding residues of Fc that are interconnected with different receptor types and thus involved in Fc-FcγR interactions and recognition. Traditionally, optimisation of therapeutic antibodies has focused on mutations that exhibit increased binding to the activating receptor fcyriii [ U.S. Pat. No. 6,737,056] or reduced affinity to fcyriib [ US2009/0010920A1 ]. In all these alternative variants, mutations are introduced in both strands simultaneously.

Monoclonal antibodies often exhibit their therapeutic activity by inducing spatial localization of the target and effector immune cells. Natural antibodies mediate this by interacting with targets using their Fab domains and effector cells using Fc domains. They are capable of juxtaposing immune complexes and effector cells face-to-face so that cell-mediated responses can be induced. The avidity of antibodies required for fcγr signaling, which arises from the formation of immune complexes involving the targeting of multiple antibody molecules to a single target, is another example of the importance of spatiotemporal organization of immunization.

There are also spatiotemporal aspects to cell signaling induced as part of the effector activity of mAb molecules. Cell signaling such as those based on activation of fcγr molecules involves localization of the relevant receptor molecules within a region of the membrane domain known as a lipid raft. Lipid rafts are rich in glycosphingolipids and cholesterol and several classes of upstream signaling molecules, including Src family kinases. Following cell stimulation, various signaling molecules, adapter proteins, and signaling kinases and phosphatases are recruited. Molecular assembly at lipid rafts is important for signal transduction.

The combination of different antigen specificities and increased affinities to provide better binding properties is the basis for bispecific therapeutic design, a non-natural design strategy. Bispecific antibodies or other forms of bifunctional or multifunctional protein therapeutics are designed to mediate interactions between targets and various effector cells [ Muller & Kontermann (2010) BioDrugs (2): 89-98]. The multispecific therapeutic molecules are engineered to re-target helper T cells or other immune effector cells to specific target cells.

In another embodiment, the invention relates to a method for identifying an Fc variant polypeptide on a computer chip based on calculated binding affinities to fcyriia, fcyriib, and/or fcyriiia. In another embodiment, the method further comprises calculating the electrostatic, solvation, packaging density, hydrogen binding and entropy effects of the Fc variant polypeptide on a computer chip. In yet another embodiment, the methods of the invention further comprise constructing an Fc variant polypeptide and expressing the polypeptide in a mammalian cell in a therapeutic antibody setting and further expressing the antibody. In yet another embodiment, the method of the present invention comprises: fc variant polypeptides identified on a computer chip were constructed by site-directed mutagenesis, PCR-based mutagenesis, cassette mutagenesis, or de novo synthesis.

Factors considered in the design of synthetic Fc scaffolds include the calculation of steric exclusion, changes in buried interface regions, relative contact density, relative solvation, and electrostatic effects on a computer chip. All of these matrices were used to achieve affinity scores.

In one aspect, the present application describes molecular designs that achieve a fine fcγr selectivity profile via designing asymmetric scaffolds built on heterodimeric fcs. The scaffold allows asymmetric mutations in the CH2 domain to achieve a variety of new selectivity profiles. In addition, scaffolds have inherent features for engineering multifunctional (bi-, tri-, tetra-or penta-functional) therapeutic molecules.

In certain embodiments, the asymmetric scaffold may be optimized for pH-dependent binding properties to the neonatal Fc receptor (FcRn) to enable better recycling of the molecule and enhance its half-life and associated pharmacokinetic properties.

Asymmetric scaffolds can be optimized for binding of functionally related fcyri receptor allotypes. Fcyri is a major marker on macrophages involved in chronic inflammatory disorders such as rheumatoid arthritis, atopic dermatitis, psoriasis and various lung diseases.

Asymmetric scaffolds can be optimized for protein a binding. Protein a binding is often used to isolate and purify antibody molecules. Mutations can be introduced into the asymmetric scaffold to avoid aggregation of the therapeutic agent during storage.

Accordingly, it is specifically contemplated that Fc variants of the invention may contain, inter alia, one or more additional amino acid residue substitutions, mutations and/or modifications that result in antibodies having preferred characteristics, including, but not limited to: increased serum half-life, increased binding affinity, reduced immunogenicity, increased yield, enhanced or reduced ADCC and CDC activity, altered glycosylation and/or disulfide bonds and modified binding specificity.

It is contemplated that Fc variants of the invention may have other altered properties relative to comparable molecules, including increased in vivo half-life (e.g., serum half-life) in mammals, particularly humans, increased stability and/or increased melting temperature (Tm) in vivo (e.g., serum half-life) and/or in vitro (e.g., shelf life). In one embodiment, the Fc variants of the invention have the following in vivo half-lives: greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. In another embodiment, the Fc variants of the invention have the following in vitro half-lives (e.g., liquid or powder formulations): greater than 15 days, greater than 30 days, greater than 2 months, greater than 3 months, greater than 6 months, or greater than 12 months, or greater than 24 months, or greater than 36 months, or greater than 60 months.

It will also be appreciated by those skilled in the art that the Fc variants of the invention may have altered immunogenicity when administered to a subject. Accordingly, modified CH3 domains that minimize immunogenicity of Fc variants can be considered generally more desirable for therapeutic applications.

The Fc variants of the invention may be combined with other Fc modifications, including but not limited to modifications that alter effector function. The invention includes combining the Fc variants of the invention with other Fc modifications to provide additive, synergistic, or novel properties in antibodies or Fc fusion proteins. Such modification may be in the hinge, CH1, or CH2, (or CH 3) domain, provided that it does not negatively alter the stability and purity characteristics of the modified CH3 domains of the invention, or a combination thereof. It is contemplated that the Fc variants of the invention enhance the characteristics of the modifications combined with them. For example, if an Fc variant of the invention is combined with a mutant (which is known to bind fcγriiia with higher affinity than a comparable molecule comprising a wild-type Fc region); then combination with the mutants of the invention results in a higher fold enhancement in fcyriiia affinity.

In one embodiment, the Fc variants of the invention may be combined with other known Fc variants, such as those disclosed in the following references: duncan et al 1988,Nature 332:563-564; lund et al 1991,J Immunol 147:2657-2662; lund et al 1992,Mol Immunol 29:53-59; alegre et al, 1994, transformation 57:1537-1543; hutchins et al, 1995,Proc Natl.Acad Sci USA 92:11980-11984; jefferis et al, 1995,Immunol Lett.44:111-117; lund et al 1995,Faseb J9:115-119; jefferis et al, 1996,Immunol Lett 54:101-104; lund et al 1996,Immunol 157:4963-4969; armour et al 1999,Eur J Immunol 29:2613-2624; idusogie et al 2000,J Immunol 164:4178-4184; reddy et al 2000,J Immunol 164:1925-1933; xu et al 2000,Cell Immunol 200:16-26; idusogie et al 2001,J Immunol 166:2571-2575; shields et al 2001,J Biol Chem 276:6591-6604; jefferis et al, 2002,Immunol Lett 82:57-65; presta et al 2002,Biochem Soc Trans 30:487-490); U.S. Pat. nos. 5,624,821;5,885,573;6,194,551; U.S. patent publication nos. 60/601,634 and 60/608,852; PCT publication Nos. WO00/42072 and WO 99/58372.

Those of skill in the art will appreciate that the Fc variants of the invention may have altered ligand (e.g., fcγr, C1 q) binding characteristics of Fc (examples of binding characteristics include, but are not limited to, binding specificity, equilibrium dissociation constant (K _D), dissociation and binding rate (K _off and K _on, respectively), binding affinity, and/or antibody antigenicity), and that certain alterations are more or less desirable. It is well known in the art that the equilibrium dissociation constant (K _D) is defined as K _off/k_on. It is generally understood that binding molecules (e.g., and antibodies) having a low K _D are more preferred than binding molecules (e.g., and antibodies) having a high K _D. However, in some cases, the K _on or K _off values may be more correlated than the K _D values. One skilled in the art can determine which kinetic parameters are most important for a given antibody application. For example, a modified CH3 and/or CH2 that enhances Fc binding to one or more positive modulators (e.g., fcγriiia) while not altering or even reducing Fc binding to the negative modulator fcγriib would be more advantageous for enhancing ADCC activity. Alternatively, modified CH3 and/or CH2 that reduces binding to one or more positive modulators and/or enhances binding to fcyriib would be advantageous for reducing ADCC activity. Accordingly, the ratio of binding affinities (e.g., equilibrium dissociation constant (K _D)) may indicate whether ADCC activity of the Fc variant is enhanced or reduced. For example, a decrease in the ratio of fcyriiia/fcyriib equilibrium dissociation constants (K _D) will correlate with improved ADCC activity, while an increase in the ratio will correlate with a decrease in ADCC activity.

As part of the characterization of Fc variants, their binding affinities to fcyriiia (CD 16 a) and fcyriib (CD 32 b) were tested, reported as ratios compared to wild-type IgG 1. (see example 4 and Table 5) in such cases, it is possible to evaluate the effect of CH3 binding domain mutations on binding to these activating and inhibiting Fc receptors. In one embodiment, provided herein is an isolated heteromultimer comprising a heteromultimeric Fc region, wherein the heteromultimeric Fc region comprises a modified CH3 domain having increased stability, the modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70 ℃, wherein the binding of the heterodimer to CD16a is about the same as compared to the wild-type homodimer. In certain embodiments, the binding of the heterodimer to CD16a is increased as compared to the wild-type homodimer. In alternative embodiments, the binding of the heterodimer to CD16a is reduced compared to the wild-type homodimer.

In certain embodiments, provided herein are isolated heteromultimers comprising a heteromultimeric Fc region, wherein the heteromultimeric Fc region comprises a modified CH3 domain having increased stability, the modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) greater than 70 ℃, wherein the binding of the heterodimer to CD32b is about the same as compared to the wild-type homodimer. In certain embodiments, the binding of the heterodimer to CD32b is increased as compared to the wild-type homodimer. In alternative embodiments, the binding of the heterodimer to CD32b is reduced compared to the wild-type homodimer.

Those skilled in the art will appreciate that instead of reporting K _D binding to CD16a and CD32b as the ratio of Fc variants to wild-type homodimers, K _D can be reported as the ratio of Fc variants to CD16a binding to Fc variants to CD32b (data not shown). This ratio may provide an indication of modified CH3 domain mutation versus wild type for ADCC or unchanged, or increased or decreased, as described in more detail below.

The affinity and binding properties of the Fc variants of the invention for fcγr are initially determined using in vitro assays known in the art for determining Fc-fcγr interactions, i.e., specific binding of the Fc region to fcγr (biochemical or immunological based assays), including but not limited to ELISA assays, surface plasmon resonance assays, immunoprecipitation assays (see section entitled "characterization and functional assays" below), and other methods such as indirect binding assays, competitive inhibition assays, fluorescence Resonance Energy Transfer (FRET), gel electrophoresis, and chromatography (e.g., gel filtration). These and other methods may utilize labels on one or more components being detected and/or utilize various detection methods including, but not limited to, chromogenic, fluorescent, luminescent, or isotopic labels. For a detailed description of binding affinity and kinetics see Paul, W.E. code, fundamental Immunology, 4 th edition, lippincott-Raven, philadelphia (1999), which focuses on antibody-immunogen interactions.

It is contemplated that the binding properties of the molecules of the invention are also characterized by an in vitro functional assay for determining the function of one or more fcγr mediator effector cells (see section entitled "characterization and functional assay" below). In certain embodiments, the molecules of the invention have binding properties in vivo models (e.g., those described and disclosed herein) similar to those of in vitro-based assays. However, the invention does not exclude molecules of the invention that do not exhibit the desired phenotype in an in vitro based assay, but exhibit the desired phenotype in vivo.

The present invention encompasses Fc variants that bind fcyriiia (CD 16 a) with increased affinity relative to a comparable molecule. In particular embodiments, the Fc variants of the invention bind fcyriiia with increased affinity and fcyriib (CD 32 b) with unchanged or reduced binding affinity relative to a comparable molecule. In yet another embodiment, the Fc variants of the invention have a reduced ratio of fcyriiia/fcyriib equilibrium dissociation constants (K _D) relative to a comparable molecule.

The present invention also contemplates Fc variants that bind fcγriiia (CD 16 a) with reduced affinity relative to a comparable molecule. In particular embodiments, the Fc variants of the invention bind fcyriiia with reduced affinity relative to a comparable molecule, and bind fcyriib with unchanged or increased binding affinity relative to a comparable molecule.

In one embodiment, the Fc variant binds fcyriiia with increased affinity. In particular embodiments, the Fc variant has an affinity for fcyriiia that is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least 80-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold higher than that of the comparable molecule. In other embodiments, the affinity of the Fc variant for fcyriiia is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least S0%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule.

In another embodiment, the equilibrium dissociation constant (K _D) of an Fc variant for an Fc ligand (e.g., fcγ R, C.sup.1 q) is reduced between about 2-fold and 10-fold, or between about 5-fold and 50-fold, or between about 25-fold and 250-fold, or between about 100-fold and 500-fold, or between about 250-fold and 1000-fold, relative to a comparable molecule.

In another embodiment, the Fc variant has at least a 2-fold, or at least a 3-fold, or at least a 5-fold, or at least a 7-fold, or at least a 10-fold, or at least a 20-fold, or at least a 30-fold, or at least a 40-fold, or at least a 50-fold, or at least a 60-fold, or at least a 70-fold, or at least a 80-fold, or at least a 90-fold, or at least a 100-fold, or at least a 200-fold, or at least a 400-fold, or at least a 600-fold decrease in the equilibrium dissociation constant (K _D) for fcγriiia relative to a comparable molecule. In another embodiment, the equilibrium dissociation constant (K _D) of the Fc variant for fcγriiia is reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule.

In one embodiment, the Fc variant binds fcyriib with constant or reduced affinity. In particular embodiments, the affinity of the Fc variant for fcyriib is unchanged or reduced by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold relative to a comparable molecule. In other embodiments, the affinity of the Fc variant for fcyriib is unchanged or reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule.

In another embodiment, the Fc variant has an equilibrium dissociation constant (K _D) for fcγriib that is unchanged or increased by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 30-fold, or at least 40-fold, or at least 50-fold, or at least 60-fold, or at least 70-fold, or at least S0-fold, or at least 90-fold, or at least 100-fold, or at least 200-fold relative to a comparable molecule. In another particular embodiment, the equilibrium dissociation constant (K _D) of the Fc variant for fcyriib is unchanged or increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule.

In yet another embodiment, the Fc variant binds fcyriiia with increased affinity relative to a comparable molecule and fcyriib with unchanged or decreased binding affinity relative to a comparable molecule. In particular embodiments, the Fc variant has an increase in affinity for fcyriiia of at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold relative to a comparable molecule. In another particular embodiment, the affinity of the Fc variant for fcyriib is either unchanged or reduced by at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold relative to a comparable molecule. In other embodiments, the affinity of the Fc variant for fcyriiia is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule and the affinity of the Fc variant for fcyriib is unchanged or increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule.

In yet another embodiment, the ratio of the Fc variant to the fcyriiia/fcyriib equilibrium dissociation constant (K _D) of a comparable molecule is reduced. In particular embodiments, the ratio of the Fc variant relative to the fcyriiia/fcyriib equilibrium dissociation constant (K _D) of a comparable molecule is reduced by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold. In another particular embodiment, the ratio of the Fc variant relative to the fcyriiia/fcyriib equilibrium dissociation constant (K _D) of a comparable molecule is reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200%.

In another embodiment, the Fc variant binds fcyriiia with reduced affinity relative to a comparable molecule. In particular embodiments, the affinity of the Fc variant for fcyriiia is reduced by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold relative to a comparable molecule. In other embodiments, the affinity of the Fc variant for fcyriiia is reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule.

In yet another embodiment, the Fc variant binds fcyriiia with reduced affinity and fcyriib with constant or increased affinity relative to a comparable molecule. In particular embodiments, the Fc variant has at least a 1-fold, or at least a 3-fold, or at least a 5-fold, or at least a 10-fold, or at least a 20-fold, or at least a 50-fold, or at least a 100-fold decrease in affinity for fcyriiia relative to a comparable molecule. In another particular embodiment, the Fc variant has at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold higher affinity for fcyriib than a comparable molecule. In other embodiments, the affinity of the Fc variant for fcyriiia is reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule and the affinity of the Fc variant for fcyriib is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule.

In yet another embodiment, the Fc variant increases the equilibrium dissociation constant (K _D) for fcyriiia by at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold when compared to a comparable molecule. In particular embodiments, the Fc variant has at least a 2-fold, or at least a 3-fold, or at least a 5-fold, or at least a 7-fold, or at least a 10-fold, or at least a 20-fold, or at least a 50-fold, or at least a 100-fold decrease in the equilibrium dissociation constant (K _D) for fcγriib relative to a comparable molecule.

CH2 variation for fcγR selectivity

The Fc-fcγr protein-protein interactions in this complex indicate that the two chains in the Fc molecule interact with two different sites on the fcγr molecule. Despite symmetry in the two heavy chains in a natural Fc molecule, the local fcγr environment near a residue on one chain differs from the fcγr residue around the same residue position on the opposite Fc chain. Two symmetrically related positions interact with different choices of fcγr residues.

Given the asymmetry in Fc binding to fcγr, simultaneous mutations in chains a and B of the Fc molecule do not affect interactions with fcγr in a symmetrical manner. When mutations are introduced to optimize interactions on one chain of the Fc with its local fcγr environment, the corresponding mutations in the second chain in the homodimeric Fc structure may be advantageous, detrimental or unassisted for the desired fcγr binding and selectivity profile.

Using structural and computationally guided methods, asymmetric mutations were engineered in both chains of Fc to overcome these limitations of traditional Fc engineering strategies that introduce identical mutations on both chains of Fc. Better binding selectivity between receptors can be obtained if both chains of Fc are independently optimized for enhanced binding to their corresponding faces of the receptor molecule.

For example, mutations at specific positions on one chain of an Fc can be designed to enhance selectivity for specific residues, which is a positive design effort, while the same residue positions can be mutated to interact adversely with their local environment in alternative fcγ receptor types, which is a negative design effort, to achieve better selectivity between the two receptors. In certain embodiments, methods are provided for designing asymmetric amino acid modifications in the CH2 domain that selectively bind one fcγ receptor compared to a different fcγ receptor (e.g., selectively bind FcgRIIIa instead of FcgRIIb). In other certain embodiments, methods are provided for designing asymmetric amino acid modifications in the CH2 domain of variant Fc heterodimers, comprising amino acid modifications in the CH3 domain to promote heterodimer formation. In another embodiment, methods are provided for designing selectivity for different fcγ receptors based on variant Fc heterodimers comprising asymmetric amino acid modifications in the CH2 domain. In yet another embodiment, methods for designing asymmetric amino acid modifications that favor binding of an fcγ receptor to one side of an Fc molecule are provided. In other certain embodiments, methods are provided for designing polar drivers that favor fcγ receptors for interaction with only one side of a variant Fc heterodimer comprising asymmetric amino acid modifications in the CH2 domain.

The asymmetric design of mutations in the CH2 domain can be tailored to recognize fcγr on one side of the Fc molecule. This constitutes the generating face of the asymmetric Fc scaffold, while the opposite face exhibits a wild-type interaction-prone pattern without a designed selectivity profile and can be considered as a non-generating face. A negative design strategy can be employed to introduce mutations on the non-generating face to block fcγr interactions with the face of the asymmetric Fc scaffold where fcγreceptors have a desired propensity for interaction by forcing them.

Table E: possibly interesting selectivity profile of Fc for different fcγ receptors

(. Sup.ll.) shows variants that exhibit increased or wild-type binding to a specific receptor type or one of its isoforms. (×) shows no apparent binding to receptor or subset allotypes.

The invention also relates to fusion polypeptides comprising a binding domain fused to an Fc region, wherein the Fc region comprises a modified CH3 domain having increased stability, the modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) above 70 ℃. It is specifically contemplated that molecules comprising heterodimers comprising modified CH3 domains may be generated by methods well known to those skilled in the art. Briefly, these methods include, but are not limited to, combining a variable region or binding domain having a desired specificity (e.g., a variable region isolated from a phage display or expression library, or derived from a human or non-human antibody or binding domain of a receptor) with a variant Fc heterodimer. Alternatively, one skilled in the art can generate variant Fc heterodimers by modifying the CH3 domain in the Fc region of a molecule (e.g., an antibody) comprising the Fc region.

In one embodiment, the Fc variant is an antibody or Fc fusion protein. In a particular embodiment, the invention provides an antibody comprising an Fc region comprising a modified CH3 domain with increased stability, the modified CH3 domain comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) above 70 ℃. Such antibodies include IgG molecules that can be modified to produce Fc variants that naturally contain an Fc region comprising a CH3 domain, or antibody derivatives that have been engineered to contain an Fc region comprising a modified CH3 domain. The Fc variants of the invention include any antibody molecule that binds, preferably specifically binds (i.e., competes for non-specific binding as determined by immunoassays well known in the art for determining specific antigen-antibody binding) an antigen comprising an Fc region that incorporates a modified CH3 domain. Such antibodies include, but are not limited to, polyclonal, monoclonal, monospecific, bispecific, multispecific, human, humanized, chimeric antibodies, single chain antibodies, fab fragments, F (ab') ₂ fragments, disulfide-linked Fvs, and Complementarity Determining Regions (CDRs) containing VL or VH domains, even specifically binding antigens, in some cases fragments engineered to contain or fuse to variant Fc heterodimers.

"Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to the binding of secreted antibodies to Fc receptors (fcrs) on certain cytotoxic cells, such as Natural Killer (NK) cells, neutrophils and macrophages, so that these cytotoxic effector cells specifically bind to antigen-bearing target cells, which are then killed by the cytotoxin in their cytotoxic form. Specific high affinity IgG antibodies targeting the target cell surface "arm" the cytotoxic cells, which is absolutely required for this killing effect. Target cell lysis is an intracellular process that requires direct intercellular contact and does not involve complement.

The ability of any particular antibody to mediate target cell lysis by ADCC can be assayed. To evaluate ADCC activity, an antibody of interest is added to a target cell along with an immune effector cell, which can be activated by an antigen-antibody complex, thereby causing cell lysis of the target cell. Cell lysis is typically detected by lysing the cells to release a label (such as a radioactive substrate, fluorescent dye, or native intracellular protein). Effector cells that may be used in such assays include Peripheral Blood Mononuclear Cells (PBMC) and Natural Killer (NK) cells. Specific examples of in vitro ADCC assays are found in WISECARVER et al, 1985, 79:277; bruggemann et al 1987,J Exp Med 166:1351; wilkinson et al 2001,J Immunol Methods 258:183; patel et al 1995J Immunol Methods 184:29 and described herein (see section entitled "characterization and functional assays" below). Alternatively or in addition, the ADCC activity of the antibody of interest can be assessed in vivo, for example in an animal model as described in Clynes et al, 1998,PNAS USA 95:652.

It is contemplated that the Fc variants of the invention are characterized by an in vitro functional assay for determining effector cell function of one or more fcγr mediators. In certain embodiments, the molecules of the invention have binding properties and effector cell functions in vivo models (e.g., those described and disclosed herein) similar to those of in vitro-based assays. However, the invention does not exclude molecules of the invention that do not exhibit the desired phenotype in an in vitro based assay, but exhibit the desired phenotype in vivo.

The invention further provides Fc variants with enhanced CDC function. In one embodiment, the Fc variant has increased CDC activity. In one embodiment, the CDC activity of the Fc variant is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold greater than a comparable molecule. In another embodiment, the Fc variant binds C1q with at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 7-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold higher affinity than a comparable molecule. In yet another embodiment, the CDC activity of the Fc variant is increased by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule. In a particular embodiment, the Fc variants of the invention bind to C1q with increased affinity; has enhanced CDC activity and specifically binds to at least one antigen.

The invention also provides Fc variants with reduced CDC function. In one embodiment, the Fc variant has reduced CDC activity. In one embodiment, the CDC activity of the Fc variant is at least 2-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 50-fold, or at least 100-fold lower than a comparable molecule. In another embodiment, the Fc variant binds to C1q with at least 1-fold, or at least 3-fold, or at least 5-fold, or at least 10-fold, or at least 20-fold, or at least 50-fold, or at least 100-fold reduced affinity relative to a comparable molecule. In another embodiment, the CDC activity of an Fc variant is reduced by at least 10%, or at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or at least 100%, or at least 150%, or at least 200% relative to a comparable molecule. In certain embodiments, the Fc variant binds C1q with reduced affinity, has reduced CDC activity, and specifically binds to at least one antigen.

In some embodiments, the Fc variant comprises one or more engineered glycoforms, i.e., a glycocomposition covalently linked to a molecule comprising an Fc region. The engineered glycoforms can be used for a variety of purposes including, but not limited to, increasing or decreasing effector function. The engineered glycoforms may be produced by any method known to those skilled in the art, for example, using engineered or variant expression strains, by co-expression with one or more enzymes such as beta (1, 4) -N-acetylglucosaminyl transferase III (GnTI 11), by expression of molecules containing the Fc region in or from various organisms' cell lines, or by modification of carbohydrates following expression of molecules containing the Fc region. Methods of producing engineered sugar forms are known in the art, including but not limited to Umana et al, 1999,Nat.Biotechnol 17:176-180; davies et al 20017Biotechnol Bioeng 74:288-294; shields et al 2002,J Biol Chem 277:26733-26740; shinkawa et al, 2003,J Biol Chem 278:3466-3473); U.S. Pat. nos. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929; PCT WO 00/61739A1; PCT WO 01/292246A1; PCT WO 02/311140A1; the method described in PCT WO 02/30954A 1; potillegent ^TM technology (Biowa, inc.Princeton, N.J.); glycoMAb ^TM glycosylation engineering (GLYCART biotechnology AG, zurich, switzerland). See, for example, WO 00061739; EA01229125; US 20030115614; okazaki et al, 2004, JMB, 336:1239-49.

Contemplated Fc variants include antibodies comprising a variable region and a heterodimeric Fc region, wherein the heterodimeric Fc region comprises a modified CH3 domain with increased stability comprising an amino acid mutation that promotes heterodimer formation, wherein the modified CH3 domain has a melting temperature (Tm) above 70 ℃. Fc variants that are antibodies can be "regenerated" by combining the variable domain of a fragment thereof that specifically binds at least one antigen with a heterodimeric Fc region comprising a modified CH3 domain. Alternatively, heterodimeric Fc variants may be produced by modifying the CH3 domain of an antibody containing an Fc region that binds an antigen.

Antibodies of the invention may include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intracellular antibodies, monospecific antibodies, multispecific antibodies, bispecific antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single chain FvFc (scFvFc), single chain Fv (scFv), and anti-idiotype (anti-Id) antibodies. In particular, antibodies useful in the methods of the invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. The immunoglobulin molecules of the invention may be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igG ₁、IgG₂、IgG₃、IgG₄、IgA₁ and IgA ₂) or subclass.

Antibodies of the invention may be derived from any animal source, including birds and mammals (e.g., humans, mice, donkeys, sheep, rabbits, goats, guinea pigs, camels, horses, or chickens). In a particular embodiment, the antibody is a human or humanized monoclonal antibody, in particular a bispecific monoclonal antibody. As used herein, "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin, and include antibodies isolated from a human immunoglobulin library or from mice expressing antibodies from human genes.

Antibodies, such as all polypeptides, have an isoelectric point (pI), which is generally defined as the pH at which the polypeptide is not net charged. It is known in the art that the solubility of the protein is generally minimized when the pH of the solution is equal to the isoelectric point (pI) of the protein. It is possible to optimize solubility by varying the number and position of ionizable residues in the antibody to adjust pI. For example, the pI of a polypeptide can be manipulated by making appropriate amino acid substitutions (e.g., substitution of an uncharged residue such as alanine with a charged amino acid such as lysine). Without being bound by any particular theory, amino acid substitutions of the antibody that result in an alteration of the pI of the antibody may increase the solubility and/or stability of the antibody. The person skilled in the art will understand what amino acid substitutions are most appropriate for a particular antibody to obtain the desired pI. The pI of a protein may be determined by a variety of methods including, but not limited to: isoelectric focusing and various computer algorithms (see, e.g., bjellqvist et al, 1993,Electrophoresis 14:1023). In one embodiment, the Fc variant PI of the invention is between pH 6.2 and pH 8.0. In another embodiment, the antibody PI of the invention is between pH 6.8 and pH 7.4. In one embodiment, the substitution that results in an alteration of the pI of an Fc variant of the invention will not significantly reduce its binding affinity to an antigen. It is contemplated that modified CH3 domains with increased stability may also lead to changes in pI. In one embodiment, variant Fc heterodimers are specifically selected to affect increased stability and purity and, any desired pI changes.

Antibodies of the invention may be monospecific, bispecific, trispecific or have more specificity. The multispecific antibodies may specifically bind to different epitopes of a desired target molecule, or may specifically bind to target molecules and heterologous epitopes, such as heterologous polypeptides or solid supports. See, for example, international publication Nos. WO 94/04690, WO 93/17715, WO 92/08802, WO91/00360 and WO 92/05793; tutt et al, 1991, J.Immunol.147:60-69; U.S. Pat. nos. 4,474,893, 4,714,681, 4,925,648, 5,573,920 and 5,601,819; and Kostelny et al, 1992, J.Immunol.148:1547).

Various embodiments of multifunctional targeting molecules can be designed based on such asymmetric scaffolds as shown in figure 20.

Multispecific antibodies have binding specificities for at least two different antigens. Although such molecules typically bind only two antigens (i.e., bispecific antibodies, bsabs), the invention also includes antibodies with additional specificity, such as trispecific antibodies. Examples of bsabs include, but are not limited to, those in which one arm is directed against a tumor cell antigen and the other arm is directed against a cytotoxic molecule, or two arms are directed against two different tumor cell antigens, or two arms are directed against two different soluble ligands, or one arm is directed against a soluble ligand and the other arm is directed against a cell surface receptor, or two arms are directed against two different cell surface receptors. Methods for preparing bispecific antibodies are known in the art.

According to various methods, an antibody variable domain (antibody-antigen combining site) having a desired binding specificity is fused to an immunoglobulin constant domain sequence. May be fused to an immunoglobulin heavy chain constant region comprising at least part of the hinge, CH2 and CH3 regions. It is contemplated that the first heavy chain constant region (CH 1) containing the necessary sites for binding to the light chain is present in at least one fusion. DNA encoding the immunoglobulin heavy chain fusion and, if desired, the immunoglobulin light chain, is inserted into a separate expression vector and co-transfected into a suitable host organism. In embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields, this provides great flexibility in adjusting the mutual ratios of the three polypeptide fragments. See, example 1 and table 2. However, when expressing at least two polypeptide chains in the same ratio, resulting in high yields or the ratio is not particularly important, it is possible to insert the coding sequences of two or all three polypeptide chains into one expression vector.

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate may be conjugated to avidin and the other antibody conjugated to biotin. It is proposed to use such antibodies, for example, to target immune system cells to undesired cells (U.S. Pat. No. 4,676,980), and for the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). The heteroconjugate antibodies may be prepared using any conventional crosslinking method. Suitable crosslinking agents are well known in the art, see U.S. Pat. No. 4,676,980 and many crosslinking techniques.

Antibodies having more than two valencies and incorporating modified CH3 domains and the resulting Fc heterodimers of the invention are contemplated. For example, trispecific antibodies may be prepared. See, e.g., tutt et al, J.Immunol.147:60 (1991).

Antibodies of the invention also include antibodies having a half-life (e.g., serum half-life) in a mammal (e.g., human) of greater than 15 days, greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months. The extended half-life of an antibody of the invention in a mammal (e.g., a human) results in a higher serum titer of the antibody or antibody fragment in the mammal, thereby reducing the frequency of administration of the antibody or antibody fragment and/or reducing the concentration of administration of the antibody or antibody fragment. Antibodies with extended half-life in vitro can be produced by techniques known to those skilled in the art. For example, antibodies with increased in vivo half-life can be produced by modifying (e.g., substituting, deleting or adding) amino acid residues identified as being involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., international publication Nos. WO 97/34631; WO 04/029207; U.S. Pat. No. 6,737,056 and U.S. patent publication No. 2003/0190311).

In a particular embodiment, the variant Fc heterodimer comprising a modified CH3 domain is a multispecific antibody (referred to herein as an antibody of the invention) that specifically binds to an antigen of interest. In particular, the antibodies of the invention are bispecific antibodies. In one embodiment, the antibodies of the invention specifically bind to a polypeptide antigen. In another embodiment, the antibodies of the invention specifically bind to a non-polypeptide antigen. In yet another embodiment, administration of an antibody of the invention to a mammal suffering from a disease or disorder may result in a therapeutic benefit in the mammal.

Essentially any molecule can be targeted and/or incorporated into a variant Fc heterodimer construct (e.g., an antibody, fc fusion protein) provided herein, including, but not limited to, the following list of proteins, as well as subunits, domains, motifs and epitopes belonging to the following list of proteins: renin; growth hormone, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin a-chain; insulin B-chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VII, factor VIIIC, factor IX, tissue Factor (TF) and von willebrand factor; anticoagulants such as protein C; cardionatriuretic peptide; a pulmonary surfactant; a plasminogen activator, such as urokinase or human urine or tissue type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factors-alpha and-beta; enkephalinase; RANTES (regulated after activation, typically T cell expression and secretion); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; miao Leguan (Muellerian) -inhibitors; relaxin a-chain; relaxin B-chain; a relaxin source; a mouse gonadotropin-associated peptide; microbial proteins such as beta-lactamase; a DNase; igE; cytotoxic T-lymphocyte-associated antigens (CTLA), such as CTLA-4; inhibin; activin; vascular Endothelial Growth Factor (VEGF); receptors for hormones or growth factors, e.g., EGFR, VEGFR; interferons such as interferon-alpha (alpha-IFN), interferon-beta (beta-IFN) and interferon-gamma (gamma-IFN); protein a or D; a rheumatoid factor; neurotrophic factors such as Bone Derived Neurotrophic Factor (BDNF), neurotrophic factor-3, neurotrophic factor-4, neurotrophic factor-5 or neurotrophic factor-6 (NT-3, NT-4, NT-5 or NT-6) or nerve growth factor; platelet Derived Growth Factor (PDGF); fibroblast growth factors such as AFGF and PFGF; epidermal Growth Factor (EGF); transforming Growth Factors (TGF) such as TGF- α and TGF- β, including TGF-1, TGF-2, TGF-3, TGF-4 or TGF-5; insulin-like growth factors-I and-II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding proteins; CD proteins, such as CD2、CD3、CD4、CD8、CD11a、CD14、CD18、CD19、CD20、CD22、 CD23、CD25、CD33、CD34、CD40、CD40L、CD52、CD63、CD64、 CD80 and CD147; erythropoietin; an osteogenesis inducing factor; an immunotoxin; bone Morphogenic Proteins (BMP); interferons such as interferon- α, interferon- β and interferon- γ; colony Stimulating Factors (CSF), such as M-CSF, GM-CSF and G-CSF; interleukins (IL), such as IL-1 to IL-13; tnfα, superoxide dismutase; a T cell receptor; surface membrane proteins; decay accelerating factors; viral antigens, such as a portion of the AIDS envelope, e.g., gp120; a transfer protein; homing the recipient; address elements; regulatory proteins; cell adhesion molecules such as LFA-1, mac l, p150.95, VLA-4, ICAM-1, ICAM-3 and VCAM, a4/p7 integrin and (Xv/p 3 integrin includes any one or subunit thereof, integrin alpha subunit such as CD49a, CD49b, CD49C, CD49d, CD49e, CD49f, alpha 7, alpha 8, alpha 9, alpha D, CD a, CD11b, CD51, CD11C, CD41, alpha IIb, alpha IELb; integrin beta subunits such as CD29, CD18, CD61, CD104, beta 5, beta 6, beta 7 and beta 8, integrin subunit combinations including but not limited to alpha V beta 3, alpha V beta 5 and alpha 4 beta 7, apoptosis pathway members, igE, blood group antigens, flk2/flt3 receptors, obesity (OB) receptors, mp1 receptors, CTLA-4, protein C, eph receptors such as EphA2, ephB 4, ephB2, etc., human Leukocyte Antigens (HLA) such as HLA-DR, complement proteins such as complement receptors CR1, C1Rq and other complement factors such as C3 and C5, glycoprotein receptors such as GpIb alpha, GPIIb/IIIa and CD200, and fragments of any of the foregoing polypeptides.

Antibodies of the invention that specifically bind to cancer antigens are also provided, including but not limited to: ALK receptor (pleiotrophin receptor), pleiotrophin, KS1/4 pan-carcinoma antigen; ovarian cancer antigen (CA 125); a phosphate ester of prostanoic acid; prostate Specific Antigen (PSA); melanoma-associated antigen p97; melanoma antigen gp75; high molecular weight melanoma antigen (HMW-MAA); prostate specific membrane antigen; carcinoembryonic antigen (CEA); polymorphic epithelial mucin antigen; human milk fat globule antigen; colorectal tumor-associated antigens, such as: CEA, TAG-72, CO17-1A, GICA-9, CTA-1, and LEA; burkitt lymphoma antigen-38.13; CD19; human B-lymphoma antigen-CD 20; CD33; melanoma specific antigens such as ganglioside GD2, ganglioside GD3, ganglioside GM2 and ganglioside GM3; tumor specific graft type cell surface antigen (TSTA); viral-induced tumor antigens, including T-antigens, envelope antigens of DNA tumor viruses and RNA tumor viruses; carcinoembryonic antigen-alpha fetoproteins such as CEA of the colon, 5T4 carcinoembryonic trophoblast glycoprotein and bladder tumor carcinoembryonic antigen; differentiation antigens such as human lung cancer antigens L6 and L20; fibrosarcoma antigen; human leukemia T cell antigen-Gp 37; novel glycoproteins (neoglycoprotein); sphingolipids; breast cancer antigens such as EGFR (epidermal growth factor receptor); NY-BR-16; NY-BR-16 and HER2 antigen (p 185HER 2); polymorphic Epithelial Mucin (PEM); malignant human lymphocyte antigen-APO-1; differentiation antigens, such as the I antigen found in fetal erythrocytes; primitive endoderm I antigens found in adult human erythrocytes; embryo before implantation; i (Ma) found in gastric adenocarcinoma; m18, M39 found in mammary epithelium; SSEA-1 found in bone marrow cells; VEP8; VEP9; myl; va4-D5; d ₁ -22 found in colorectal cancer; TRA-1-85 (blood group H); SCP-1 found in testicular and ovarian cancers; c14 found in colon adenocarcinoma; f3 found in lung adenocarcinoma; AH6 found in gastric cancer; a Y hapten; ley found in embryonic cancer cells; TL5 (blood group a); EGF receptor found in A431 cells; the E ₁ series (blood group B) found in pancreatic cancer; FC10.2 found in embryonic cancer cells; gastric adenocarcinoma antigen; CO-514 (blood group Lea) found in adenocarcinomas; NS-10 found in adenocarcinoma; CO-43 (blood group Leb); a431 G49 found in the cellular EGF receptor; MH2 (blood group ALeb/Ley) found in colon adenocarcinoma; 19.9 found in colon cancer; gastric cancer mucin; t ₅A₇ found in bone marrow cells; r ₂₄ found in melanoma; 4.2, G _D3、D1.1、 OFA-1、G_M2、OFA-2、G_D2, and M1 found in embryonic cancer cells: 22:25: SSEA-3 and SSEA-4 found in 8 and 4-8 cell stage embryos; cutaneous T cell lymphoma antigen; MART-1 antigen; sialic acid Tn (STn) antigens; colon cancer antigen NY-CO-45; lung cancer antigen NY-LU-12 variant a; adenocarcinoma antigen ART1; a paraneoplastic associated brain-testis-cancer antigen (cancer neuronal antigen MA2; paraneoplastic neuronal antigen); neurotumor ventral antigen 2 (NOVA 2); hepatocellular carcinoma antigen gene 520; tumor associated antigen CO-029; tumor associated antigens MAGE-C1 (cancer/testis antigen CT 7), MAGE-B1 (MAGE-XP antigen), MAGE-B2 (DAM 6), MAGE-2, MAGE-4-a, MAGE-4-B and MAGE-X2; cancer-testis antigen (NY-EOS-1) and fragments of any of the above polypeptides.

In certain embodiments, the heteromultimers described herein comprise at least one therapeutic antibody. In some embodiments, the therapeutic antibody binds to a cancer target antigen. In one embodiment, the therapeutic antibody may be one selected from the group consisting of: abamelizumab (abagovomab), adalimumab (adalimumab), alemtuzumab, oxybutyramiumab (golimumab), papuzumab, basilizumab, beluzumab (belimumab), bevacizumab, briakinumab, kanatamab (canakinumab), cetuximab (catumaxomab), cerlizumab (certolizumab pegol), cetuximab, daclizumab, desuzumab (denosumab), efalizumab, gancicumab (galiximab), gemtuzumab ozagrimoab, golimumab (golimumab), timomumab, infliximab, irinoteb, lu Xishan, meperib, mozuab, mycograb, natalizumab, niuzumab, orelizumab (ocrelizumab), fauzumab (ofatmab), dabuzumab, oxalitumomab, ceritumomab, and other antibodies.

Antibodies of the invention include modified derivatives (i.e., covalently bound by covalent binding of any type of molecule to the antibody). For example, but not limited to, antibody derivatives include antibodies modified by methods such as glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, attachment to cellular ligands or other proteins, and the like. Any of a number of chemical modifications may be made by known techniques including, but not limited to: specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, and the like. In addition, the derivative may contain one or more non-classical amino acids.

An antibody or fragment thereof with an extended in vivo half-life can be produced by attaching a polymer molecule such as high molecular weight polyethylene glycol (PEG) to the antibody or antibody fragment. The PEG may be attached to the antibody or antibody fragment by site-specific conjugation of the PEG to the N-terminus or C-terminus of the antibody or antibody fragment, or by epsilon-amino groups present on lysine residues, with or without a multifunctional linker. Derivatization with linear or branched polymers that result in minimal loss of bioactivity may be utilized. The extent of conjugation was closely monitored by SDS-PAGE and mass spectrometry to ensure that the PEG molecules were properly conjugated to the antibodies. Unreacted PEG may be separated from the antibody-PEG conjugate by, for example, size exclusion or ion exchange chromatography.

In addition, antibodies can be conjugated to albumin to produce antibodies or antibody fragments that are more stable in vivo and have a longer in vivo half-life. Such techniques are well known in the art, see for example International publication Nos. WO 93/15199, WO 93/15200 and WO 01/77137; and European patent No. EP 413,622. The invention encompasses the use of antibodies or fragments thereof fused or conjugated to one or more moieties, including but not limited to: peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetics, synthetic drugs, inorganic molecules, and organic molecules.

The invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including covalent and non-covalent conjugation) to a heterologous protein or polypeptide (or portion thereof, e.g., a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, or at least 100 amino acids) to produce a fusion protein. Fusion need not necessarily be direct, but may be by a linker sequence. For example, antibodies may be employed to target a heterologous polypeptide to a particular cell type in vitro or in vivo by fusing or conjugating the antibody to an antibody specific for a particular cell surface receptor. Antibodies fused or conjugated to heterologous polypeptides can also be used in vitro immunoassays and purification methods using methods known in the art. See, for example, international publication No. WO 93/21232; european patent No. EP 439,095; naramura et al, 1994, immunol. Lett.39:91-99; U.S. patent No. 5,474,981; gillies et al 1992,PNAS 89:1428-1432; and Fell et al, 1991, J.Immunol.146:2446-2452.

The invention further includes compositions comprising a heterologous protein, peptide or polypeptide fused or conjugated to an antibody fragment. For example, the heterologous polypeptide may be fused or conjugated to a Fab fragment, fd fragment, fv fragment, F (ab) ₂ fragment, VH domain, VL domain, VH CDR, VL CDR, or fragment thereof. Methods of fusing or conjugating polypeptides to antibody moieties are well known in the art. See, for example, U.S. Pat. nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; european patent nos. EP 307,434 and EP 367,166; international publication Nos. WO 96/04388 and WO 91/06570; ashkenazi et al, 1991, proc. Natl. Acad. Sci. USA 88:10535-10539; zheng et al, 1995, J.Immunol.154:5590-5600; and Vil et al, 1992, proc.Natl. Acad. Sci. USA89:11337-11341.

Other fusion proteins, such as antibodies that specifically bind to an antigen (e.g., as above), can be produced by techniques of gene shuffling, motif shuffling, exon shuffling, and/or codon shuffling (collectively referred to as "DNA shuffling"). DNA shuffling may be employed to alter the activity of antibodies or fragments thereof of the present invention (e.g., antibodies or fragments thereof having higher affinity and lower dissociation rates). See, generally, U.S. Pat. nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252 and 5,837,458, and Patten et al, 1997,Curr.Opinion Biotechnol.8: 724-33; harayama,1998,Trends Biotechnol.16 (2): 76-82; hansson et al 1999, j.mol. Biol.287:265-76; and Lorenzo and blasto, 1998,BioTechniques 24 (2): 308-313. The antibody or fragment thereof, or the encoded antibody or fragment thereof, may be altered prior to recombination by error-prone PCR, random nucleotide insertion, or other methods by subjecting to random mutagenesis. One or more portions of the polynucleotide encoding an antibody or antibody fragment whose portion specifically binds an antigen may be recombined with one or more components, motifs, segments, portions, domains, fragments, etc., of one or more heterologous molecules.

The invention also includes the use of variant Fc heterodimers or fragments thereof conjugated to a therapeutic agent.

The antibody or fragment thereof may be conjugated to a therapeutic moiety, for example a cytotoxin, such as a cytostatic or cytocidal agent, a therapeutic agent, or a radioactive metal ion, such as an alpha-emitter. Cytotoxins or cytotoxic agents include any agent that is detrimental to cells. Examples include ribonucleases, monomethylauristatin E and F, paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, ipecac, mitomycin, etoposide, tenoposide (tenoposide), vincristine, vinblastine, colchicine, doxorubicin, daunomycin, dihydroxyanthrax-dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin and cyclophosphamide and their analogues or homologues. Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil, amamide), alkylating agents (e.g., mechlorethamine, thiotepa (thioepa) chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU)), cyclophosphamide (cyclophosphamide), busulfan, dibromomannitol, streptozotocin, mitomycin C and cisplatin (II) (DDP) cisplatin), anthracyclines (e.g., daunomycin and doxorubicin), antibiotics (e.g., actinomycin D (formerly actinomycin), bleomycin, mithramycin and Anthranilic (AMC)), and anti-mitogens (e.g., vincristine and vinblastine). For a more detailed list of therapeutic agents see PCT publication WO 03/075957.

In addition, antibodies and fragments thereof may be conjugated to therapeutic agents or drug moieties that alter a given biological response. The therapeutic agent or drug moiety should not be construed as being limited to classical chemotherapeutic agents only. For example, the drug moiety may be a protein or polypeptide having a desired biological activity. Such proteins may include, for example, toxins such as abrin, ricin a, ondase (or another cytotoxic rnase), pseudomonas exotoxin, cholera toxin, or diphtheria toxin; proteins such as tumor necrosis factor, interferon-alpha, interferon-beta, nerve growth factor, platelet-derived growth factor, tissue plasminogen activator, apoptotic agents such as TNF-alpha, TNF-beta, AIM I (see International publication No. WO 97/33899), AIM II (see International publication No. WO 97/34911), fas ligand (Takahashi et al, 1994, J. Immunol., 6:1567) and VEGI (see International publication No. WO 99/23105), thrombogenic agents (thrombotic agent) or anti-angiogenic agents such as angiostatin or endostatin; or biological response modifiers, for example, lymphokines (such as interleukin-1 ("IL-1"), interleukin-2 ("IL-2"), interleukin-6 ("IL-6"), granulocyte-macrophage colony-stimulating factor ("GM-CSF") and granulocyte colony-stimulating factor ("G-CSF")) or growth factors (such as growth hormone ("GH")).

Furthermore, the antibodies may be conjugated to a therapeutic moiety such as a radioactive substance or a macrocyclic chelator for conjugation to radioactive metal ions (see examples of radioactive substances above). In certain embodiments, the macrocyclic chelator is 1,4,7, 10-tetraazacyclododecane-N, N', N "-tetraacetic acid (DOTA) that may be attached to an antibody through a linker molecule. Such linker molecules are generally known in the art, see Denardo et al, 1998,Clin Cancer Res.4:2483; peterson et al 1999, bioconjug. Chem.10:553; and Zimmerman et al 1999, nucleic. Med. Biol.26:943.

Methods of fusing or conjugating antibodies to polypeptide moieties are known in the art. See, for example, U.S. Pat. nos. 5,336,603;5,622,929;5,359,046;5,349,053;5,447,851 and 5,112,946; EP 307,434; EP 367,166; PCT publications WO 96/04388 and WO 9I/06570; ashkenazi et al 1991,PNAS USA 88:10535; zheng et al 1995,J Immunol 154:5590; and Vil et al 1992,PNAS USA 89:11337. Fusion of antibodies to portions need not necessarily be direct, but may be via linker sequences. Such linker molecules are generally known in the art, see Denardo et al, 1998,Clin Cancer Res 4:2483; peterson et al 1999,Bioconjug Chem 10:553; zimmerman et al 1999,Nucl Med Biol 26:943; gamnett, 2002,Adv Drug Deliv Rev 53:17l.

Recombinant expression of an Fc variant, derivative, analog or fragment thereof (e.g., an antibody or fusion protein of the invention) requires construction of an expression vector containing a polynucleotide encoding the Fc variant (e.g., an antibody or fusion protein). Once the polynucleotide encoding the Fc variant (e.g., antibody or fusion protein) is obtained, vectors for producing the Fc variant (e.g., antibody or fusion protein) can be produced by recombinant DNA techniques using techniques well known in the art. Thus, described herein are methods of making proteins by expressing polynucleotides comprising nucleotide sequences encoding Fc variants (e.g., antibodies or fusion proteins). Expression vectors containing Fc variant (e.g., antibody or fusion protein) coding sequences and appropriate transcriptional and translational control signals can be constructed using methods well known to those skilled in the art. These methods include, for example, recombinant DNA techniques in vitro, synthetic techniques, and genetic recombination in vivo. Accordingly, the present invention provides replicable vectors comprising a nucleotide sequence encoding an Fc variant of the present invention operably linked to a promoter. Such vectors may include nucleotide sequences encoding antibody molecule constant regions (see, e.g., international publication No. WO 86/05807; international publication No. WO 89/01036; and U.S. Pat. No. 5,122,464) and antibody variable regions, or polypeptides that produce Fc variants may be cloned into such vectors for expression of full length antibody chains (e.g., heavy or light chains), or complete Fc variants comprising fusions of non-antibody derived polypeptides and Fc regions incorporating at least modified CH3 domains.

The expression vector is transferred into a host cell by conventional techniques, and the transfected cell is then cultured by conventional techniques to produce the Fc variants of the invention. Thus, the invention includes host cells comprising a polynucleotide encoding an Fc variant of the invention operably linked to a heterologous promoter. In particular embodiments, to express Fc variants containing diabodies, vectors encoding heavy and light chains may be co-expressed in host cells to express the entire immunoglobulin molecule, as described in detail below.

Various host expression vector systems may be utilized to express Fc variants (e.g., antibodies or fusion protein molecules) of the invention (see, e.g., U.S. patent No. 5,807,715). Such host expression systems represent vectors that can produce and subsequently purify the coding sequences of interest, but also represent cells that can express the Fc variants of the invention in situ when transformed or transfected with the appropriate nucleotide coding sequences. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant phage DNA, plasmid DNA, or cosmid DNA expression vectors containing Fc variant coding sequences (e.coli) and bacillus subtilis; yeast transformed with a recombinant yeast expression vector comprising an Fc variant coding sequence (e.g., pichia pastoris (Saccharomyces Pichia)); insect cell systems infected with recombinant viral expression vectors (e.g., baculovirus) containing Fc variant coding sequences; plant cell systems infected with recombinant viral expression vectors (e.g., cauliflower mosaic virus, caMV; tobacco mosaic virus, TMV) containing Fc variant coding sequences or transformed with recombinant plasmid expression vectors (e.g., ti plasmid) containing Fc variant coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NS0, and 3T3 cells) carrying recombinant expression constructs containing promoters derived from mammalian cell genomes (e.g., metallothionein promoters) or mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). In certain embodiments, bacterial cells, such as E.coli, or eukaryotic cells, are utilized to express the Fc variants as recombinant antibodies or fusion protein molecules. For example, mammalian cells such as Chinese hamster ovary Cells (CHO) in combination with vectors such as the major intermediate early gene promoter element from human cytomegalovirus are efficient expression systems for antibodies (Foecking et al, 1986, gene 45:101; and Cockett et al, 1990, bio/Technology 8:2). In a particular embodiment, the expression of a nucleotide sequence encoding an Fc variant (e.g., an antibody or fusion protein) of the invention is regulated by a constitutive promoter, an inducible promoter, or a tissue-specific promoter.

In bacterial systems, a number of expression vectors may be advantageously selected depending on the intended use of the expressed Fc variant (e.g., antibody or fusion protein). For example, where large amounts of such proteins are to be produced for use in the production of Fc variant pharmaceutical compositions, vectors may be required that direct the expression of high levels of fusion protein products that are readily purified. Such vectors include, but are not limited to, the E.coli expression vector pUR278 (Ruther et al, 1983,EMBO 12:1791), in which the Fc variant coding sequence may be ligated alone into a vector in frame with the lac Z coding region in order to produce a lac Z-fusion protein; pIN vectors (Inouye and Inouye,1985,Nucleic Acids Res.13:3101-3109; van Heeke and Schuster,1989, J.biol. Chem. 24:5503-5509); etc. pGEX vectors can also be used to express fusion proteins of foreign polypeptides with glutathione 5-transferase (GST). Such fusion proteins are generally soluble and are readily purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione. pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.

In insect systems, the noctuid (Autographa californica) nuclear polyhedrosis virus (AcNPV) was used as a vector for expression of foreign genes. The virus grows in Spodoptera frugiperda (Spodoptera frugiperda) cells. The Fc variant (e.g., antibody or fusion protein) coding sequence can be cloned separately into a non-essential region of the virus (e.g., the polyhedrin gene) and placed under the control of the AcNPV promoter (e.g., the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems are available. In the case of adenoviruses used as expression vectors, the Fc variant (e.g., antibody or fusion protein) coding sequence of interest may be linked to an adenovirus transcription/translation control complex, such as a late promoter and a tripartite leader sequence. The chimeric gene may then be inserted into the adenovirus genome by in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., the E1 or E3 region) will produce a recombinant virus that is both live and capable of expressing Fc variants (e.g., antibodies or fusion proteins) in an infected host (see, e.g., logan and Shenk,1984,Proc.Natl.Acad.Sci.USA 81:355-359). Specific initiation signals may also be required for efficient translation of the inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. In addition, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the complete insert. These exogenous translational control signals and initiation codons can be from a variety of sources, including natural and synthetic. Expression efficiency may be increased by including appropriate transcription enhancer elements, transcription terminators, and the like (see, e.g., bittner et al 1987,Methods in Enzymol.153:516-544).

Expression of an Fc variant (e.g., an antibody or fusion protein) may be controlled by any promoter or enhancer element known in the art. Promoters that may be used to control expression of genes encoding Fc variants (e.g., antibodies or fusion proteins) include, but are not limited to: SV40 early promoter region (Bernoist and Chambon,1981,Nature 290:304-310), the promoter contained in the 3' long terminal repeat of the Rous sarcoma virus (Yamamoto et al, 1980, cell 22:787-797), the herpes thymidine kinase promoter (Wagner et al, 1981, proc. Natl. Acad. Sci. U.S. A. 78:1441-1445), the regulatory sequences of metallothionein genes (Brister et al, 1982,Nature 296:39-42), the tetracycline (Tet) promoter (Gossen et al, 1995, proc. Nat. Acad. Sci. USA 89:5547-5551); prokaryotic expression vectors such as the beta-lactamase promoter (Villa-Kamaroff et al 1978, proc. Natl. Acad. Sci. U.S. A. 75:3727-3731), or the tac promoter (DeBoer et al 1983, proc. Natl. Acad. Sci. U.S. A.80:21-25; see also "Useful proteins from recombinant bacteria", publish in SCIENTIFIC AMERICAN,1980, 242:74-94); plant expression vectors containing nopaline synthase promoter regions (Herrera-Estrella et al, nature 303:209-213) or cauliflower mosaic virus 35S RNA promoter (Gardner et al, 1981,Nucl.Acids Res.9:2871) and the promoter of the photosynthesis enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al, 1984,Nature 310:115-120); promoter elements from yeasts or other fungi such as the Gal 4 promoter, the ADC (alcohol dehydrogenase) promoter, the PGK (phosphoglycerate kinase) promoter, the alkaline phosphatase promoter and the following animal transcriptional control regions, which exhibit tissue specificity and are used in transgenic animals: an elastase I gene control region active in pancreatic gland cells (Swift et al, 1984, cell 38:639-646; ornitz et al, 1986,Cold Spring Harbor Symp.Quant.Biol.50:399-409; macDonald,1987, hepatology 7:425-515); an insulin gene control region active in pancreatic beta cells (Hanahan, 1985,Nature 315:115-122), an immunoglobulin gene control region active in lymphocytes (Grosschedl et al, 1984, cell 38:647-658; adams et al, 1985,Nature 318:533-538; alexander et al, 1987, mol. Cell. Biol. 7:1436-1444), a mouse mammary tumor virus control region active in testes, breast, lymph and mast cells (Leder et al, 1986, cell 45:485-495), an albumin gene control region active in the liver (Pinkert et al, 1987,Genes and Devel.1:268-276), an alpha fetoprotein gene control region active in the liver (Krumlauf et al, 1985, mol. Cell. Biol.5:1639-1648; hammer et al, 1987,Science 235:53-58; an alpha 1-antitrypsin gene control region active in the liver (Kelsey et al, 1987,Genes and Devel.1:161-171), a beta-globin gene control region active in bone marrow cells (Mogram et al, 1985,Nature 315:338-340; kollias et al, 1986, cell 46:89-94; myelin basic protein gene control region active in oligodendrocytes of the brain (read et al, 1987, cell 48:703-712), a myosin light chain-2 gene control region active in skeletal muscle (Sani, 1985,Nature 314:283-286), a neuron-specific enolase (NSE) active in neuronal cells (Morelli et al, 1999, gen.Virol.80:571-83), a brain-derived neurotrophin (BDNF) gene control region active in neuronal cells (Tabuchi et al, 1998, biochem. Biophysical. Res. Com. 253:818-823); glial Fibrillary Acidic Protein (GFAP) promoter active in astrocytes (Gomes et al, 1999,Braz J Med Biol Res 32 (5): 619-631; morelli et al, 1999, gen. Virol. 80:571-83) and gonadotropin releasing hormone gene control region active in the hypothalamus (Mason et al, 1986,Science 234:1372-1378).

Expression vectors containing gene inserts encoding Fc variants (e.g., antibodies or fusion proteins) of the invention can be identified by three general methods: (a) nucleic acid hybridization, (b) the presence or absence of "marker" gene function, and (c) expression of the insert. In the first method, the presence or absence of a gene encoding a peptide, polypeptide, protein or fusion protein in an expression vector can be detected by nucleic acid hybridization using a probe containing a sequence homologous to an inserted gene encoding a peptide, polypeptide, protein or fusion protein, respectively. In the second approach, recombinant vector/host systems can be identified and selected based on the presence or absence of certain "marker" gene functions (e.g., thymidine kinase activity, antibiotic resistance, transformed phenotype, inclusion body formation in baculovirus, etc.) due to the insertion of nucleotide sequences encoding antibodies or fusion proteins into the vector. For example, if a nucleotide sequence encoding an Fc variant (e.g., an antibody or fusion protein) is inserted into the marker gene sequence of a vector, a recombinant containing a gene encoding an antibody or fusion protein insert can be identified by the absence of marker gene function. In a third method, the recombinant expression vector can be identified by assaying the gene product (e.g., antibody or fusion protein) expressed by the recombinant. Such assays may be based on, for example, the physical or functional properties of the fusion protein in an in vitro assay system (e.g., binding to an anti-bioactive molecule antibody).

In addition, host cell lines may be selected that modulate the expression of the insert sequence or modify and process the gene product in a particular manner desired. Expression by certain promoters may be increased in the presence of certain inducers; thus, the expression of the genetically engineered fusion protein can be controlled. Furthermore, translational and post-translational processing and modification of different host cells have characteristic and specific mechanisms (e.g., glycosylation, phosphorylation of proteins). Suitable cell lines or host systems may be selected to ensure the desired modification and processing of the expressed foreign protein. For example, expression in bacterial systems will produce products that are free of glycosylation, and expression in yeast will produce products that are glycosylated. Eukaryotic host cells may be employed that contain cellular mechanisms that possess the initial transcripts (e.g., glycosylation and phosphorylation) that properly process the gene product. Such mammalian host cells include, but are not limited to: CHO, VERY, BHK, hela, COS, MDCK, 293, 3T3, WI38, NS0, in particular neuronal cell lines such AS SK-N-AS, SK-N-FI, SK-N-DZ human neuroblastoma (Sugimoto et al 1984,J.Natl.Cancer Inst.73:51-57), SK-N-SH human neuroblastoma (Biochim. Biophys. Acta,1982, 704:450-460), daoy human cerebellum (He et al, 1992,Cancer Res.52:1144-1148) DBTRG-05MG glioblastoma cells (Kruse et al, 1992,In Vitro Cell.Dev.Biol.28A:609-614), IMR-32 human neuroblastoma (Cancer Res.,1970, 30:2110-2118), 1321N1 human astrocytoma (Proc. Natl. Acad. Sci. USA,1977, 74:4816), MOG-G-CCM human astrocytoma (Br. J. Cancer,1984, 49:269), U87MG human glioblastoma-astrocytoma (Acta Pathol. Microbiol. Scand.,1968, 74:465-486), A172 human glioblastoma (Olopade et al, 1992,Cancer Res.52:2523-2529), C6 rat glioblastoma cells (Benda et al, 1968,Science 161:370-371), neuro-2a mouse neuroblastoma (Proc.Natl. Acad. Sci. USA,1970, 65:129-136), NB41A3 mouse neuroblastoma (Proc.Natl. Acad. Sci. USA,1962, 48:4-1190), ovine choroid plexus (Bolin et al, 1994,J.Virol.Methods 48:211-221), G355-5, PG-4 cat normal astrocytes (Haapala et al, 1985, J. Virol. 53:827-833), mpf ferret al (Trowbridge et al, 1982,In Vitro 18:952-960) and normal cell lines, for example, CTX TNA2 rats have normal cortex (Radany et al, 1992,Proc.Natl.Acad.Sci.USA 89:6467-6471), such as CRL7030 and Hs578Bst. In addition, different vector/host expression systems can affect processing reactions to varying degrees.

For long-term, high-yield production of recombinant proteins, stable expression is often preferred. For example, cell lines that stably express an Fc variant (e.g., an antibody or fusion protein) of the invention may be engineered. Rather than using expression vectors containing viral origins of replication, host cells are transformed with DNA and selectable markers controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylation sites, etc.). After introduction of the foreign DNA, the engineered cells are grown in the nutrient rich medium for 1-2 days and then switched to selective medium. The selectable marker in the recombinant plasmid confers resistance to selection so that the cell can stably integrate the plasmid into its chromosome, grow to form a foci, and can then clone and expand into a cell line. This approach can be advantageously used to engineer cell lines that express Fc variants that specifically bind to antigens. Such engineered cell lines are particularly useful in screening and evaluating compounds that affect the activity of Fc variants (e.g., polypeptides or fusion proteins) that specifically bind to an antigen.

Many selection systems can be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, 1977, cell 11:223), hypoxanthine-guanine phosphoribosyl transferase (Szybalska and Szybalski,1962,Proc.Natl.Acad.Sci.USA 48:2026), and adenine phosphoribosyl transferase (Lowy et al, 1980, cell 22:817) genes, which can be used in tk-cells, hgprt-cells, or aprt-cells, respectively. In addition, antimetabolite resistance may be used as a basis for selection dhfr, gpt, neo and hygro genes, dhfr conferring methotrexate resistance (Wigler et al, 1980,Natl.Acad.Sci.USA 77:3567;O'Hare et al, 1981,Proc.Natl.Acad.Sci.USA 78:1527); gpt confers mycophenolic acid resistance (Mulligan and Berg,198l,Proc.Natl.Acad.Sci.USA 78:2072); neo confers aminoglycoside G-418 resistance (Colberre-Garapin et al, 1981, J.mol. Biol. 150:1); hygro confers hygromycin resistance (Santerre et al, 1984, gene 30:147).

Once the Fc variant (e.g., antibody or fusion protein) is produced by recombinant expression, it can be purified by any method of protein purification known in the art, such as chromatography (e.g., ion exchange chromatography, affinity chromatography, particularly affinity chromatography for a particular antigen after use of protein a, and size column chromatography), centrifugation, differential solubility, or any other standard protein purification technique.

The Fc variant is typically recovered from the culture medium as a secreted polypeptide, although it may also be recovered from the host cell lysate when no secretion signal is directly produced. If the Fc variant is membrane-bound, it can be released from the membrane using a suitable detergent solution (e.g., triton-X100).

When the Fc variant is produced in a recombinant cell other than one of human origin, it is completely free of human-derived proteins or polypeptides. However, it is necessary to purify the Fc variant from recombinant cellular proteins or polypeptides to obtain a substantially homogeneous formulation for the Fc variant. As a first step, the culture medium or lysate is typically centrifuged to remove particulate cell debris.

The Fc heterodimer with the antibody constant regions can be conveniently purified by hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography (affinity chromatography is a preferred purification technique). Depending on the polypeptide to be recovered, other protein purification techniques such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica, chromatography on heparin sepharose, chromatography on anion or cation exchange resins such as polyaspartic acid columns, chromatofocusing, SDS-PAGE and ammonium sulfate precipitation may also be used. The suitability of protein a as an affinity ligand depends on the type and isotype of immunoglobulin Fc domain used. Protein A can be used to purify immunoglobulin Fc regions based on the human gamma 1, gamma 2, or gamma 4 heavy chain (Lindmark et al J.Immunol. Meth.62:1-13 (1983)). G protein is recommended for all mouse subtypes and for human gamma 3 (Guss et al, EMBO J.5:15671575 (1986)). The matrix to which the affinity ligand binds is most commonly agarose, but other matrices may be used. Physically stable substrates such as controlled pore glass or poly (styrene divinyl) benzene can achieve faster flow rates and shorter processing times than agarose. The conditions used to bind the immunoadhesion molecules to the protein a or G affinity column depend entirely on the characteristics of the Fc domain; i.e. its species and isotype. Generally, when the appropriate ligand is selected, efficient binding occurs directly from unconditioned broth. Bound variant Fc heterodimers can be eluted efficiently at acidic pH (3.0 or above 3.0), or in neutral pH buffers containing mild chaotropic salts. This affinity chromatography step can result in a variant Fc heterodimer preparation that is >95% pure.

Expression levels of Fc variants (e.g., antibodies or fusion proteins) can be increased by vector amplification (for review see Bebbington and Hentschel,The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning,, volume 3 (ACADEMIC PRESS, new York, 1987)). For example, when the markers in the vector system expressing the antibody or fusion protein are amplified, an increase in the level of inhibitor present in the host cell culture will increase the copy number of the marker gene. Since the amplified region is linked to an antibody gene, the yield of the antibody or fusion protein will also be increased (Crouse et al, 1983, mol. Cell. Biol. 3:257).

Two expression vectors of the invention can be used to cotransfect a host cell. For example, a first vector encodes a heavy chain derived polypeptide and a second vector encodes a light chain derived polypeptide. The two vectors may contain the same selectable marker, enabling equivalent expression of the heavy and light chain polypeptides. Alternatively, a single vector encoding and capable of expressing the fusion protein or the heavy and light chain polypeptides may be used. The coding sequences for the fusion protein or heavy and light chains may comprise cDNA or genomic DNA.

Characterization and functional assays

The Fc variants (e.g., antibodies or fusion proteins) of the invention can be characterized in various ways. In one embodiment, the purity of the variant Fc heterodimer is assessed using techniques well known in the art, including but not limited to SDS-PAGE gels, western blots, densitometry, or mass spectrometry. Protein stability may be characterized using a range of techniques, not limited to size exclusion chromatography, UV visible and CD spectroscopy, mass spectrometry, differential light scattering, bench top stability assays, freeze thawing plus other characterization techniques, differential scanning calorimetry, differential scanning fluorometry, hydrophobic interaction chromatography, isoelectric focusing, receptor binding assays, or relative protein expression levels. In one exemplary embodiment, the stability of the modified CH3 domain is assessed by the melting temperature of the variant Fc heterodimer as compared to the wild-type CH3 domain using techniques well known in the art, such as differential scanning calorimetry or differential scanning fluorometry.

The ability of the Fc variants of the invention to specifically bind to a ligand (e.g., fcγriiia, fcγriib, C1 q) can also be determined. Such assays can be performed in solution (e.g., houghten, bio/Techniques,13:412-421,1992), on beads (Lam, nature,354:82-84, 1991, on chip (Fodor, nature,364:555-556,1993), on bacteria (U.S. Pat. No. 5,223,409), on plasmids (Cull et al, proc. Natl. Acad. Sci. USA,89:1865-1869,1992), or on phages (Scott and Smith, science,249:386-390,1990; devlin, science,249:404-406,1990; cwirla et al, proc. Natl. Acad. Sci. USA,87:6378-6382,1990; and Felici, J. Mol. Biol.,222:301-310,1991).

Specific binding of the Fc variants of the invention to a molecule such as an antigen (e.g., a cancer antigen and cross-reactivity with other antigens) or ligand (e.g., fcγr) can be determined by any method known in the art. Immunoassays that can be used to analyze specific binding and cross-reactivity include, but are not limited to, competitive and non-competitive assay systems using the following techniques: such as western blot, radioimmunoassay, ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassays, immunoprecipitation assays, precipitant reactions, gel diffusion precipitant reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluoroimmunoassay, protein A immunoassays, to name a few. These assays are conventional and well known in the art (see, e.g., ausubel et al, edit 1994,Current Protocols in Molecular Biology, 1 st edition, john Wiley & Sons, inc., new York).

The binding affinity and the rate of dissociation of interactions of the Fc variants of the invention with molecules, such as antigens or ligands (e.g., fcγr), can be determined by competitive binding assays. One example of a competitive binding assay is a radioimmunoassay that includes incubating a labeled ligand, such as fcγr (e.g., 3H or 125I), with a molecule of interest (e.g., an Fc variant of the invention) in the presence of increasing amounts of unlabeled ligand, such as fcγr, and detecting the molecule that binds to the labeled ligand. Affinity and binding dissociation rates of the molecules of the invention for the ligand can be determined from the saturation data by scatchard analysis.

Kinetic parameters of Fc variants can also be determined using any Surface Plasmon Resonance (SPR) based assay known in the art (e.g., BIAcore kinetic analysis). For a review based on SPR technology see Mullet et al, 2000, methods 22:77-91; dong et al 2002,Review in Mol.Biotech, 82:303-23; fivash et al, 1998,Current Opinion in Biotechnology 9:97-101; rich et al 2000,Current Opinion in Biotechnology 11:54-61. Furthermore, in U.S. patent No. 6,373,577;6,289,286; 5,322,798;5,341,215;6,268,125 any SPR instrument and SPR-based method for measuring protein-protein interactions described in 6,268,125 are encompassed within the methods of the present invention.

Fluorescence Activated Cell Sorting (FACS) using any technique known to those skilled in the art can be used to characterize the binding of Fc variants to molecules expressed on the cell surface (e.g., fcyriiia, fcyriib). The flow sorter is able to rapidly examine a large number of individual cells (e.g., 10,000,000-100,000,000 cells/hr) containing library inserts (shape et al, PRACTICAL FLOW, cytometric, 1995). Flow cytometry for sorting and examining biological cells is well known in the art. Known flow cytometers are described, for example, in U.S. patent nos. 4,347,935, 5,464,581, 5,483,469, 5,602,039, 5,643,796, and 6,211,477. Other known flow cytometers are FACS VANTAGE ^TM systems manufactured by Becton Dickinson and Company, and COPAS ^TM systems manufactured by Union biomerica.

The Fc variants of the invention may be characterized by their Fc variant-mediated effector cell function. Examples of effector cell functions that can be assayed include, but are not limited to, antibody-dependent cell-mediated cytotoxicity (ADCC), phagocytosis, opsonization, opsonophagocytosis (opsonophagocytosis), C1q binding, and complement-dependent cell-mediated cytotoxicity (CDC). Any cell-based or cell-free assay known to those of skill in the art for determining effector cell functional activity may be used (for effector cell assays, see Perussia et al, 2000,Methods Mol.Biol.121:179-92; baggiolini et al, 1998Experientia,44 (10): 841-8; lehmann et al, 2000J. Immunol. Methods,243 (1-2): 229-42;Brown E J.1994,Methods Cell Biol, 45:147-64; munn et al, 1990J. Exp. Med.,172:231-237, abdul-Majid et al, 2002Scand. J. Immunol.55:70-81; ding et al, 1998,Immunity 8:403-411).

In particular, the FcgammaR-mediated ADCC activity of the Fc variants of the present invention can be assayed in effector cells (e.g., natural killer cells) using any standard method known to those of skill in the art (see, e.g., perussia et al, 2000,Methods Mol.Biol.121:179-92). An exemplary assay for determining ADCC activity of a molecule of the invention is based on a 51Cr release assay comprising: labelling target cells with [51cr ] na ₂CrO₄ (this cell membrane permeable molecule is generally used for labelling because it binds to cytoplasmic proteins, which are released spontaneously from cells with slow kinetics but in large amounts after necrosis of the target cells); conditioning target cells with the Fc variants of the invention; combining the conditioned radiolabeled target cells and effector cells in a suitable target cell to effector cell ratio on a microtiter plate; culturing the mixture of cells at 37℃for 16-18 hours; collecting supernatant; and the radioactivity was analyzed. Cytotoxicity of the molecules of the invention can then be determined, for example, using the following formula: lysis% = (experimental cpm-target leakage cpm)/(detergent lysis cpm-target leakage cpm) xl00%. Alternatively, lysis% = (ADCC-AICC)/(maximum release-spontaneous release). Specific cleavage can be calculated using the following formula: specific cleavage = cleavage of the molecule of the invention% -cleavage in the absence of the molecule of the invention. By altering the target cell: the ratio of effector cells or antibody concentration can be mapped.

Methods for characterizing the ability of Fc variants to bind C1q and mediate Complement Dependent Cytotoxicity (CDC) are well known in the art. For example, to determine C1q binding, a C1q binding ELISA may be performed. Exemplary assays may include the following: assay plates were coated with polypeptide variants or starter polypeptides (control) in coating buffer overnight at 4C. The plates may then be washed and blocked. After washing, an aliquot of human C1q was added to each well and incubated for 2 hours at room temperature. After further washing, 100uL of sheep anti-complement C1q peroxidase conjugated antibody can be added to each well and incubated for 1 hour at room temperature. The plate can be washed again with wash buffer and 100uL of substrate buffer containing OPD (O-phenylenediamine dihydrochloride (Sigma)) added to each well. The oxidation reaction observed by the appearance of yellow may be allowed to proceed for 30 minutes and stopped by the addition of 100ul 4.5nh2so4. The absorbance can then be read at (492-405) nm.

To assess complement activation, a Complement Dependent Cytotoxicity (CDC) assay (e.g., as described in Gazzano-Santoro et al, 1996,J.Immunol.Methods 202:163) may be performed. Briefly, various concentrations of Fc variants and human complement can be diluted with buffer. Cells expressing the antigen to which the Fc variant binds may be diluted to a density of about 1 x 106 cells/ml. The mixture of Fc variants, diluted human complement, and antigen expressing cells can be added to flat bottom tissue culture 96-well plates, allowing incubation at 37C, 5% co2 for 2 hours to promote complement-mediated cell lysis. 50uL of alma blue (Accumed International) can then be added to each well and incubated overnight at 37C. Absorbance was measured with a 96-well fluorometer with excitation at 530nm and emission at 590 nm. The results can be expressed in Relative Fluorescence Units (RFU). Sample concentrations can be calculated from a standard curve, reporting percent activity relative to a comparable molecule (i.e., a molecule comprising an Fc region with an unmodified or wild-type CH3 domain) for the Fc variant of interest.

Complement assays can be performed with guinea pig, rabbit or human serum. Complement lysis of target cells can be detected by monitoring intracellular enzymes such as Lactate Dehydrogenase (LDH), as described in Korzeniewski et al, 1983,Immunol.Methods 64 (3): 313-20; and Decker et al 1988,J.Immunol Methods 115 (1): 61-9; or wherein the target cell is labeled with an intracellular label such as europium, chromium 51 or indium 111.

Method of

The invention includes administering one or more Fc variants (e.g., antibodies) of the invention to an animal, particularly a mammal, particularly a human, to prevent, treat or ameliorate one or more symptoms associated with a disease, disorder or infection. The Fc variants of the invention are particularly useful for treating or preventing diseases or conditions in which altered efficacy of effector cell function (e.g., ADCC, CDC) is desired. Fc variants and compositions thereof are particularly useful in the treatment or prevention of primary or metastatic neoplastic diseases (i.e., cancers) and infectious diseases. The molecules of the invention may be provided in pharmaceutically acceptable compositions known in the art or as described herein. As described in detail below, the molecules of the invention may be used in methods of treating or preventing cancer (particularly passive immunotherapy), autoimmune diseases, inflammatory disorders, or infectious diseases.

The Fc variants of the invention may also be advantageously used in combination with other therapeutic agents known in the art to treat or prevent cancer, autoimmune diseases, inflammatory disorders, or infectious diseases. In a particular embodiment, the Fc variants of the present invention can be used in combination with monoclonal or chimeric antibodies, lymphokines or hematopoietic growth factors (e.g., IL-2, IL-3 and IL-7) to increase the number or activity of effector cells that interact with the molecule and to increase the immune response. The Fc variants of the invention may also be advantageously used in combination with one or more drugs for the treatment of diseases, disorders or infections, including, for example, anticancer, anti-inflammatory or antiviral drugs.

Accordingly, the present invention provides methods for preventing, treating, or ameliorating one or more symptoms associated with cancer and related conditions by administering one or more Fc variants of the invention. While not wishing to be bound by any mechanism of action, the Fc variants of the invention (which bind fcyriiia and/or fcyriia with higher affinity than comparable molecules and further bind fcyriib with lower affinity than comparable molecules) and/or have enhanced effector functions, e.g., ADCC, CDC, phagocytosis, opsonization, etc.) will result in selective targeting and efficient destruction of cancer cells.

The invention also includes the administration of one or more Fc variants of the invention in combination with other therapies known to those of skill in the art to treat or prevent cancer, including but not limited to: currently standard and experimental chemotherapy, hormonal therapy, biological therapy, immunotherapy, radiation therapy or surgery. In some embodiments, the molecules of the invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more anti-cancer drugs, therapeutic antibodies, or other drugs known to those skilled in the art for treating and/or preventing cancer. Examples of dosing regimens and treatments that may be used with the Fc variants of the invention are well known in the art, see, for example, PCT publication nos. WO 02/070007 and WO 03/075957 for details.

Cancers and related conditions that may be treated or prevented with the methods and compositions of the present invention include, but are not limited to: leukemia, lymphoma, multiple myeloma, bone and connective tissue sarcoma, brain tumor, breast cancer, adrenal cancer, thyroid cancer, pancreatic cancer, pituitary cancer, eye cancer, vaginal cancer, vulvar cancer, cervical cancer, uterine cancer, ovarian cancer, esophageal cancer, gastric cancer, colon cancer, rectal cancer, liver cancer, gall bladder cancer, bile duct cancer, lung cancer, testicular cancer, prostate cancer, and penile cancer; oral cancer, salivary gland cancer, throat cancer, skin cancer, kidney cancer, bladder cancer (for a review of these conditions see Fishman et al, 1985, medicine, 2 nd edition, J.B. Lippincott Co., philadelphia and Murphy et al) ,1997,Informed Decisions：The Complete Book of Cancer Diagnosis,Treatment,and Recovery,Viking Penguin,Penguin Books U.S.A.,Inc.,United States of America).

The invention further contemplates engineering any antibody known in the art for use in the treatment and/or prevention of cancer and related disorders such that the antibody comprises an Fc region that incorporates a modified CH3 domain of the invention.

In a particular embodiment, a molecule of the invention (e.g., comprising a variant Fc heterodimer) inhibits or reduces the growth of a primary tumor or metastasis of a cancer cell by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% as compared to the growth or metastasis of a primary tumor in the absence of the molecule of the invention.

The invention includes the use of one or more Fc variants of the invention for preventing, treating or controlling one or more symptoms associated with an inflammatory disorder in a subject. While not wishing to be bound by any mechanism of action, fc variants with enhanced affinity for fcyriib will result in inhibition of activated receptors and thus immune responses, and therapeutic efficacy for the treatment and/or prevention of autoimmune disorders. Furthermore, antibodies that bind to more than one target associated with an inflammatory disorder, such as bispecific antibodies comprising variant Fc heterodimers, may provide a potentiator effect relative to monovalent therapy.

The invention also includes the administration of an Fc variant of the invention in combination with a therapeutically or prophylactically effective amount of one or more anti-inflammatory agents. The invention also provides a method of preventing, treating or managing one or more symptoms associated with an autoimmune disease, further comprising administering to the subject an Fc variant of the invention in combination with a therapeutically or prophylactically effective amount of one or more immunomodulatory agents. Examples of autoimmune diseases that can be treated by administering the Fc variants of the invention include, but are not limited to: alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune addison's disease, autoimmune anemia of the adrenal gland, autoimmune hemolysis, autoimmune hepatitis, autoimmune oophoritis and orchitis, autoimmune thrombocytopenia, behcet's disease, bullous pemphigoid, cardiomyopathy, steatorrhea-dermatitis, chronic Fatigue Immune Dysfunction Syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, cumulus-schiff syndrome, cicatricial pemphigoid, CREST syndrome, condensed set disease, crohn's disease, discoid lupus, idiopathic mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis, graves' disease, guillain-Barre syndrome (Guillain-Barre), hashimoto's thyroiditis, idiopathic pulmonary fibrosis, idiopathic Thrombocytopenic Purpura (ITP) IgA neuropathy, juvenile arthritis, lichen planus, lupus erythematosus, meniere's disease, mixed connective tissue disease, multiple sclerosis, type 1 or immune-mediated diabetes, myasthenia gravis, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, multiple chondritis, polyadenylic syndrome, polymyalgia rheumatica, polymyositis and dermatomyositis, primary agalobemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, raynaud's phenomenon (raynaud's phenomenons), lyter's syndrome, rheumatoid arthritis, sarcoidosis, scleroderma, sjogren's syndrome, systemic myotonic syndrome, systemic lupus erythematosus, large arteritis, temporal arteritis/giant cell arteritis, ulcerative colitis, uveitis, vasculitis such as dermatitis herpetiformis vasculitis, vitiligo and wegener granulomatosis. Examples of inflammatory disorders include, but are not limited to: asthma, encephalitis, inflammatory bowel disease, chronic Obstructive Pulmonary Disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathies, undifferentiated arthropathy, arthritis, inflammatory osteolysis and chronic inflammation due to chronic viral or bacterial infection. Some autoimmune disorders are associated with inflammatory diseases, and thus, overlap is considered between autoimmune and inflammatory disorders. Thus, some autoimmune disorders may be characterized as inflammatory disorders. Examples of inflammatory conditions that may be prevented, treated or controlled in accordance with the methods of the invention include, but are not limited to: asthma, encephalitis, inflammatory bowel disease, chronic Obstructive Pulmonary Disease (COPD), allergic disorders, septic shock, pulmonary fibrosis, undifferentiated spondyloarthropathies, undifferentiated arthropathy, arthritis, inflammatory osteolysis and chronic inflammation due to chronic viral or bacterial infection.

The Fc variants of the invention may also be used to reduce inflammation in animals, particularly mammals, suffering from inflammatory conditions. In a particular embodiment, the Fc of the invention reduces inflammation in an animal by at least 99%, at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, at least 50%, at least 45%, at least 40%, at least 45%, at least 35%, at least 30%, at least 25%, at least 20%, or at least 10% as compared to the inflammation in an animal not administered the molecule.

The invention further contemplates engineering any antibody known in the art for use in the treatment and/or prevention of an autoimmune disease or inflammatory disorder such that the antibody comprises a variant Fc heterodimer of the invention.

The invention also includes a method of treating or preventing an infectious disease in a subject, the method comprising administering a therapeutically or prophylactically effective amount of one or more Fc variants of the invention. Infectious diseases that can be treated or prevented with the Fc variants of the invention are caused by infectious agents including, but not limited to, viruses, bacteria, fungi, protozoa, and viruses.

Viral diseases that can be treated or prevented with the Fc variants of the invention in conjunction with the methods of the invention include, but are not limited to, diseases caused by: hepatitis A virus, hepatitis B virus, hepatitis C virus, influenza virus, varicella virus, adenovirus, herpes simplex virus type I (HSV-I), herpes simplex virus type II (HSV-II), bovine epidemic virus, rhinovirus, echovirus, rotavirus, respiratory syncytial virus, papilloma virus, papovavirus, cytomegalovirus, echinovirus, arbovirus, hantavirus (hunta virus), coxsackie virus, mumps virus, measles virus, rubella virus, polio virus, smallpox virus, EB virus, human immunodeficiency virus type I (HIV-I), human immunodeficiency virus type II (HIV-II), viral diseases such as viral meningitis, encephalitis, dengue or smallpox.

Bacterial diseases caused by bacteria that can be treated or prevented with the Fc variants of the invention and the methods of the invention include, but are not limited to: mycoplasmosis, neisseria, streptococcus pneumoniae (s. Pneumonia) disease, borrelia brucei (Borrelia burgdorferi) disease (lyme disease), bacillus anthracis (Bacillus antracis) disease (anthrax), tetanus, streptococcal disease, staphylococcal disease, mycobacteriosis, tetanus, pertussis, cholera, plague, diphtheria, chlamydia disease, staphylococcus aureus (s. Aureus) disease and legionella. Protozoal diseases that may be treated or prevented with the molecules of the invention and the methods of the invention include, but are not limited to: leishmaniasis, kestosis (kokzidioa), trypanosomiasis or malaria. Parasitic diseases caused by parasites that can be treated or prevented with the molecules of the invention and methods of the invention include, but are not limited to: chlamydia and rickettsiosis.

In some embodiments, the Fc variants of the invention may be administered in combination with a therapeutically or prophylactically effective amount of one or more other therapeutic agents known to those skilled in the art for the treatment and/or prophylaxis of infectious diseases. The present invention contemplates the use of the molecules of the present invention in combination with other molecules known to those skilled in the art for the treatment and/or prevention of infectious diseases, including but not limited to antibiotics, antifungals, and antivirals.

The invention provides methods and pharmaceutical compositions comprising the Fc variants (e.g., antibodies, polypeptides) of the invention. The invention also provides methods of treating, preventing, and ameliorating one or more symptoms associated with a disease, disorder, or infection by administering to a subject an effective amount of at least one Fc variant of the invention, or a pharmaceutical composition comprising at least one Fc variant of the invention. In one aspect, the Fc variants are substantially purified (i.e., substantially free of substances that limit their effects or produce adverse side effects, including homodimers and other cellular materials). In a particular embodiment, the subject is an animal, such as a mammal, including a non-primate (e.g., cow, pig, horse, cat, dog, rat, etc.) and a primate (e.g., monkey, such as macaque and human). In a particular embodiment, the subject is a human. In yet another particular embodiment, the antibodies of the invention are from the same species as the subject.

The route of administration of the composition depends on the disease being treated. For example, intravenous injection may be preferred for the treatment of systemic disorders such as lymphoma or tumors that have metastasized. The skilled artisan can determine the dosage of the composition to be administered without undue experimentation in combination with standard dose response studies. Relevant environmental factors considered in making those determinations include the disease being treated, the choice of composition to be administered, the age, weight and response of the individual patient, and the severity of the patient's symptoms. Depending on the circumstances, the composition may be administered to the patient by oral, parenteral, intranasal, vaginal, rectal, lingual, sublingual, buccal and/or transdermal routes.

Thus, without undue experimentation, the compositions may be designed for oral, lingual, sublingual, buccal or buccal administration by means well known in the art, for example with inert diluents or with edible carriers. The composition may be encapsulated in a gelatin capsule or compressed into tablets. For the purpose of oral therapeutic administration, the pharmaceutical composition of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing agents and the like.

Tablets, pills, capsules, troches and the like may also contain binders, acceptors (disintegrants), lubricants, sweeteners and/or flavouring agents. Some examples of binders include microcrystalline cellulose, gum tragacanth and gelatin. Examples of excipients include starch and lactose. Some examples of disintegrants include alginic acid, corn starch, and the like. Examples of lubricants include magnesium stearate and potassium stearate. An example of a glidant is colloidal silicon dioxide. Some examples of sweeteners include sucrose, saccharin and the like. Examples of flavoring agents include peppermint, methyl salicylate, orange flavoring, and the like. The materials used to prepare these various compositions should be pharmaceutically pure and non-toxic at the levels used.

The pharmaceutical compositions of the present invention may be administered by parenteral routes, such as intravenous, intramuscular, intrathecal and/or subcutaneous injection. Parenteral administration may be carried out by incorporating the compositions of the present invention into a solution or suspension. Such solutions or suspensions may also contain sterile diluents such as water for injection, saline solutions, fixed oils, polyethylene glycols, glycerol, propylene glycol and/or other synthetic solvents. Parenteral formulations may also contain antibacterial agents such as benzyl alcohol and/or methylparaben, antidotes such as ascorbic acid and/or sodium bisulphite, and chelating agents such as EDTA. Buffers such as acetates, citrates and phosphates, as well as tonicity adjusting agents such as sodium chloride and dextrose may also be added. Parenteral formulations may be enclosed in ampules, disposable syringes, and/or multiple dose vials made of glass or plastic. Rectal administration includes administration of the composition to the rectum and/or large intestine. Such administration may be accomplished using suppositories and/or enemas. Suppositories can be prepared by methods known in the art. Transdermal administration includes percutaneous absorption of the composition through the skin. Transdermal formulations include patches, ointments, creams, gels, ointments and the like. The compositions of the present invention may be administered to a patient nasally. As used herein, nasal administration or nasal administration includes administration of the composition to the nostrils and/or mucous membranes of the nasal cavity of a patient.

The pharmaceutical compositions of the present invention may be used according to the methods of the present invention for preventing, treating or ameliorating one or more symptoms associated with a disease, disorder or infection. It is contemplated that the pharmaceutical compositions of the invention are sterile and in a suitable form for administration to a subject.

In one embodiment, the compositions of the present invention are pyrogen-free formulations that are substantially free of endotoxin and/or related pyrogen-related substances. Endotoxins include toxins that are confined within microorganisms and are released upon destruction or death of the microorganism. Pyrogens also include heat-inducing thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. If administered to humans, these substances can cause fever, hypotension and shock. Due to the potentially detrimental effects, it is advantageous to remove even small amounts of endotoxin from the intravenously administered drug solution. The food and drug administration ("FDA") has specified that in intravenous administration applications, the upper limit per hour is 5 Endotoxin Units (EU)/dose/kg body weight (The United States Pharmacopeial Convention, pharmacopeial Forum (1): 223 (2000)). When therapeutic proteins are administered in amounts of hundreds or thousands of milligrams per kilogram of body weight, as is the case with monoclonal antibodies, removal of even trace amounts of endotoxin is advantageous. In particular embodiments, the endotoxin and pyrogen levels in the composition are less than 10EU/mg, or less than 5EU/mg, or less than 1EU/mg, or less than 0.1EU/mg, or less than 0.01EU/mg, or less than 0.001EU/mg.

The present invention provides methods of preventing, treating or ameliorating one or more symptoms associated with a disease, disorder or infection, the method comprising: (a) Administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of a composition comprising one or more Fc variants, and (b) subsequently administering one or more doses of said Fc variants to maintain the plasma concentration of Fc variants at a desired level (e.g., about 0.1-100 μg/ml), which continuously binds antigen. In a particular embodiment, the plasma concentration of the Fc variant is maintained at 10 μg/ml, 15 μg/ml, 20 μg/ml, 25 μg/ml, 30 μg/ml, 35 μg/ml, 40 μg/ml, 45 μg/ml or 50 μg/ml. In a particular embodiment, the effective amount of the Fc variant to be administered is at least 1mg/kg to 8mg/kg per dose. In another specific embodiment, the effective amount of the Fc variant to be administered is at least 4mg/kg to 5mg/kg per dose. In yet another particular embodiment, the effective amount of the Fc variant to be administered is from 50mg to 250mg per dose. In yet another specific embodiment, the effective amount of the Fc variant to be administered is from 100mg to 200mg per dose.

The invention also includes a regimen for preventing, treating or ameliorating one or more symptoms associated with a disease, disorder or infection, wherein the Fc variant is used in combination with a treatment (e.g., a prophylactic or therapeutic agent) other than the Fc variant and/or variant fusion protein. The invention is based in part on the following recognition: the Fc variants of the invention are capable of potentiating the effects, synergising, increasing the effectiveness, increasing the tolerability and/or reducing the side effects of other cancer treatments, including existing standard therapies and experimental chemotherapies. The combination therapies of the invention have additive efficacy, additive therapeutic effect or synergistic effect. The combination therapies of the invention enable the prevention, treatment or amelioration of one or more symptoms associated with a disease, disorder or infection with a low dose therapy (e.g., a prophylactic or therapeutic agent) in combination with an Fc variant, and/or the administration of such prophylactic or therapeutic agents to a subject suffering from a disease, disorder or infection at a lower frequency to improve the quality of life of the subject and/or to achieve a prophylactic or therapeutic effect. In addition, the combination therapies of the invention reduce or avoid adverse or deleterious side effects associated with the administration of existing monotherapy and/or existing combination therapies, thereby increasing patient compliance with the treatment regimen. Many molecules that can be used in conjunction with the Fc variants of the invention are well known in the art. See, for example, PCT publication WO 02/070007, WO 03/075957 and U.S. patent publication 2005/064514.

The present invention provides kits comprising in one or more containers one or more Fc variants having altered binding affinity to fcγr and/or C1q and altered ADCC and/or CDC activity that specifically bind to an antigen, conjugated or fused to a detectable agent, therapeutic agent or drug for monitoring, diagnosing, preventing, treating or ameliorating one or more symptoms associated with a disease, disorder or infection.

For the purpose of example only, the invention includes but is not limited to the following technical solutions:

technical scheme 1 an isolated heterodimeric Fc construct comprising a modified heterodimeric CH3 domain comprising:

A first modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide, and a second modified CH3 domain polypeptide comprising at least three amino acid modifications compared to a wild-type CH3 domain polypeptide;

Wherein at least one of the first and second CH3 domain polypeptides comprises an amino acid modification of K392J, wherein J is selected from L, I or an amino acid having a side chain volume that is not substantially greater than K;

Wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 74 ℃ and a purity of at least 95%; and is also provided with

Wherein at least one amino acid modification is not a modification of an amino acid at the interface between the first and the second CH3 domain polypeptides.

Claim 2. The isolated heterodimeric Fc construct of claim 1 comprising at least one T350X modification, wherein X is a natural or unnatural amino acid selected from valine, isoleucine, leucine, methionine, and derivatives or variants thereof.

Technical solution 3. The isolated heterodimeric Fc construct of claim 1, comprising at least one T350V modification.

The isolated heterodimeric Fc construct of any one of claims 1-3, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater.

Technical solution 5 the isolated heterodimeric Fc construct according to claim 4 wherein said modified CH3 domain has a Tm of about 77 ℃ or higher.

The isolated heterodimeric Fc construct of any one of claims 4-5, wherein the modified CH3 domain has a Tm of about 80 ℃ or greater.

Claim 7 the isolated heterodimeric Fc construct of any one of claims 1-6, wherein the at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of L351, F405, and Y407.

Claim 8 the isolated heterodimeric Fc construct of any one of claims 1-7, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of T366.

The isolated heterodimeric Fc construct according to any one of claims 1-8, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions L351, F405, and Y407, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising amino acid modifications at positions T366, K392, and T394.

Technical solution the isolated heterodimeric Fc construct of claim 9, wherein the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V and the second CH3 domain polypeptide comprises amino acid modifications T366L, K392M and T394W.

Technical solution the isolated heterodimeric Fc construct of claim 9, wherein the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V and the second CH3 domain polypeptide comprises amino acid modifications T366L, K392L and T394W.

Technical solution the isolated heterodimeric Fc construct of claim 9, wherein the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V and the second CH3 domain polypeptide comprises amino acid modifications T366I, K392M and T394W.

Technical solution the isolated heterodimeric Fc construct of claim 9, wherein the first CH3 domain polypeptide comprises amino acid modifications L351Y, F a and Y407V and the second CH3 domain polypeptide comprises amino acid modifications T366I, K392L and T394W.

The isolated heterodimeric Fc construct of any one of claims 1-11, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position S400.

Technical scheme 15. The isolated heterodimeric Fc construct of claim 12, comprising the modification S400Z, wherein Z is selected from the group consisting of a positively charged amino acid and a negatively charged amino acid.

The isolated heterodimeric Fc construct according to any one of claims 1-13, wherein said first CH3 domain polypeptide comprises an amino acid modification selected from the group consisting of S400E and S400R.

The isolated heterodimeric Fc construct of any one of claims 1-11, wherein at least one of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide comprising an amino acid modification at position N390.

The isolated heterodimeric Fc construct of claim 15, comprising the modification N390Z, wherein Z is selected from the group consisting of a positively charged amino acid and a negatively charged amino acid.

The isolated heterodimeric Fc construct of any one of claims 1-16, wherein the second CH3 domain comprises the amino acid modification N390R.

The isolated heterodimeric Fc construct of any one of claims 1-17, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification S400E and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising the amino acid modification N390R.

The isolated heterodimeric Fc construct of any one of claims 1-18, wherein each of the first and second CH3 domain polypeptides is a modified CH3 domain polypeptide, one of the modified CH3 domain polypeptides comprising the amino acid modification Q347R and the other of the modified CH3 domain polypeptides comprising the amino acid modification K360E.

The isolated heterodimeric Fc construct of any one of claims 1-4, wherein the at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising an amino acid modification of at least one of K409 and T411.

Claim 23 the isolated heterodimeric Fc construct of claim 22 comprising at least one of K409F, T E and T411D.

The isolated heterodimeric Fc construct of any one of claims 1-23, wherein at least one CH3 domain polypeptide is a modified CH3 domain polypeptide comprising a D399 amino acid modification.

Claim 25 the isolated heterodimeric Fc construct of claim 24 comprising at least one of D399R and D399K.

The isolated heterodimeric Fc construct of any one of claims 1-25, wherein the first CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of K409F, T E and T411D, and the second CH3 domain polypeptide is a modified CH3 domain polypeptide comprising at least one amino acid modification selected from the group consisting of Y407A, Y I, Y407V, D399R and D399K.

The isolated heterodimeric Fc construct of any one of claims 1-26, comprising a first modified CH3 domain comprising one of the amino acid modifications T366V, T366I, T366A, T M and T366L; and a second modified CH3 domain comprising the amino acid modification L351Y.

The isolated heterodimeric Fc construct of any one of claims 1-27, comprising a first modified CH3 domain comprising one of the amino acid modifications K392L or K392E; and a second modified CH3 domain comprising one of the amino acid modifications S400R or S400V.

An isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least four amino acid mutations,

Wherein at least one of said first and said second modified CH3 domain polypeptides comprises a mutation selected from the group consisting of N390Z and S400Z, wherein Z is selected from the group consisting of a positively charged amino acid and a negatively charged amino acid, and

Wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 70 ℃ and a purity of at least 90%,

The isolated heterodimeric Fc construct of claim 30, comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, each modified CH3 domain polypeptide comprising at least three amino acid mutations,

Wherein one of said first and said second modified CH3 domain polypeptides comprises a mutation selected from the group consisting of T411E and T411D, and

Wherein the first and second modified CH3 domain polypeptides preferentially form heterodimeric CH3 domains having a melting temperature (Tm) of at least about 70 ℃ and a purity of at least 90%.

The isolated heterodimer according to any one of claims 29-30, wherein the first modified CH3 domain polypeptide comprises an amino acid modification at positions F405 and Y407 and the second modified CH3 domain polypeptide comprises an amino acid modification at position T394.

Claim 32. The isolated heterodimer of claim 31, wherein the first modified CH3 domain polypeptide comprises an amino acid modification at position L351.

Claim 33 the isolated heterodimer of claim 31, wherein the second modified CH3 domain polypeptide comprises a modification at least one of positions T366 and K392.

The isolated heterodimer of any one of claims 29-34, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ and is formed in a purity of at least about 95%.

The isolated heterodimer of any one of claims 29-34, wherein the at least one modified CH3 domain polypeptide comprises an amino acid modification of at least one of N390R, S E and S400R.

The isolated heterodimer of any one of claims 29-35, wherein one of the first and second modified CH3 domain polypeptides comprises an amino acid modification at position 347 and the other modified CH3 domain polypeptide comprises an amino acid modification at position 360.

The isolated heterodimer of any one of claims 29-36, wherein at least one of the first and second modified CH3 domain polypeptides comprises an amino acid modification of T350V.

The isolated heterodimer according to one of claims 29-37, wherein the first modified CH3 domain polypeptide comprises at least one amino acid modification selected from the group consisting of L351Y, F a and Y407V; and the second modified CH3 domain polypeptide comprises at least one amino acid modification selected from T366L, T366I, K392L, K392M and T394W.

Claim 39 the isolated heterodimer according to any one of claims 29-30, wherein the first modified CH3 domain polypeptide comprises amino acid modifications at positions D399 and Y407 and the second modified CH3 domain polypeptide comprises amino acid modifications at positions K409 and T411.

Claim 40. The isolated heterodimer of claim 39, wherein the first CH3 domain polypeptide comprises an amino acid modification at position L351 and the second modified CH3 domain polypeptide comprises amino acid modifications at positions T366 and K392.

Technical solution 41. The isolated heterodimer of claim 40, wherein at least one of the first and second CH3 domain polypeptides comprises an amino acid modification of T350V.

The isolated heterodimer according to any one of claims 39-41, wherein the modified CH3 domain has a melting temperature (Tm) of at least about 75 ℃ or greater and is formed in a purity of at least about 95%.

Claim 43 the isolated heterodimer of claim 39, wherein the first modified CH3 domain polypeptide comprises an amino acid modification selected from the group consisting of L351Y, D399R, D399K, S400D, S400E, S400R, S K, Y407A and Y407V; and the second modified CH3 domain polypeptide comprises an amino acid modification selected from the group consisting of T366V, T366I, T366L, T366M, N390D, N390 42392E, K L, K392I, K D, K E, K409F, K409W, T D and T411E.

Technical scheme 44 an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366I, K392M and T394W.

An isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366I, K392L and T394W.

Technical scheme 46 an isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366L, K392M and T394W.

An isolated heterodimeric Fc construct comprising a modified CH3 domain comprising a first modified CH3 domain polypeptide comprising amino acid modifications L351Y, F a and Y407V; and a second modified CH3 domain polypeptide comprising amino acid modifications T366L, K392L and T394W.

Technical solution 48 the isolated heterodimeric Fc construct of any one of claims 1-47, comprising a modified Fc region based on a G-type immunoglobulin (IgG).

Technical solution 49 the isolated heterodimeric Fc construct of technical solution 48, wherein the IgG is one of IgG2 and IgG 3.

Technical solution 50 the isolated heterodimeric Fc construct of any one of claims 1-47, comprising an immunoglobulin M (IgM) -based modified Fc region.

Technical solution 51 the isolated heterodimeric Fc construct of any one of claims 1-47, comprising an immunoglobulin a (IgA) -based modified Fc region.

Technical solution 52 the isolated heterodimeric Fc construct according to any one of claims 1-47 comprising an immunoglobulin D (IgD) -based modified Fc region.

Technical solution 53 the isolated heterodimeric Fc construct according to any one of claims 1-47 comprising an immunoglobulin E (IgE) -based modified Fc region.

The isolated heterodimeric Fc construct of any one of claims 1-49, wherein the heterodimer is a bispecific antibody.

Technical solution 55 the isolated heterodimeric Fc construct of any one of claims 1-49, wherein the heterodimer is a multispecific antibody.

Claim 56 a composition comprising the isolated heterodimeric Fc construct of any one of claims 1-55 and a pharmaceutically acceptable carrier.

Claim 57. A mammalian host cell comprising a nucleic acid encoding the isolated heterodimeric Fc construct according to any one of claims 1-55.

Technical solution 58 the isolated heterodimeric Fc construct of any one of claims 1-55, comprising at least one therapeutic antibody.

The isolated heterodimeric Fc construct of any one of claims 1-55, wherein the heterodimer comprises at least one therapeutic antibody selected from the group consisting of: abamelizumab, adalimumab, alemtuzumab, ox Luo Gelei, bapidizumab, basilizumab, belimumab, bevacizumab, briakinumab, kanagamab, cetuximab, daclizumab, desuzumab, efalizumab, gancicumab, gemtuzumab oxzomib, golimumab, timox, infliximab, ipilimumab, lu Xishan, mepolimumab, motuzumab, moruzumab, mycograb, natalizumab, nimuzumab, oreuzumab, oxuzumab, maltuzumab, panitumumab, ceruzumab, rezuzumab, rituximab, tiuzumab, tolizumab, tozumab/timab, toxazumab, fauzumab, toxab, toxalizumab and toxalizumab.

Technical solution 60 a method of treating cancer in a patient having cancer characterized by a cancer antigen, the method comprising administering to the patient a therapeutically effective amount of the isolated heterodimeric Fc construct according to any one of claims 1-59.

Technical scheme 61. A method of treating an immune disorder in a patient suffering from an immune disorder characterized by an immune antigen, the method comprising administering to the patient a therapeutically effective amount of an isolated heterodimeric Fc construct according to any one of claims 1-59.

Examples

The following examples are given to illustrate the practice of the invention. They are not intended to limit or define the full scope of the invention.

Example 1: production of bivalent monospecific antibodies with heterodimeric Fc domains.

Genes encoding heavy and light chains of antibodies were constructed via gene synthesis using codons optimized for human/mammalian expression. Fab sequences were generated from known Her2/neu binding abs (Carter p. Et al (1992)Humanization of an anti P185 Her2 antibody for human cancer therapy.Proc Natl Acad Sci 89,4285.) and Fc is IgG1 isotype (SEQ ID NO: 1.) the final gene product was subcloned into the mammalian expression vector pTT5 (NRC-BRI,Canada)(Durocher,Y.,Perret,S.&Kamen,A.High-level and high-throughput recombinant protein production by transient transfection of suspension-growing human HEK293-EBNA1 cells. Nucleic acids research 30,E9(2002)). to introduce mutations in the CH3 domain via site-directed mutagenesis of the pTT5 template vector for a list of modified CH3 domain mutations see tables 1 and 6 and 7.

To estimate the formation of heterodimers and to determine the ratio of homodimers compared to heterodimers, two heterodimer heavy chains with different sizes of C-terminal extension were designed (specifically, a chain a with His tag at the C-terminal and a chain B with mRFP at the C-terminal plus strepptagii). This difference in molecular weight makes the homodimer different from the heterodimer in non-reducing SDS-PAGE, as shown in fig. 25A.

HEK293 cells were transfected with 1mg/mL 25kDa polyethylenimine (PEI, polysciences) at a PEI: DNA ratio of 2.5:1 in exponential growth phase (1.5 to 2 million cells/mL). (Raymond C. Et al A simplified polyethylenimine-mediated transfection process for large-scale and high-throughput applications.Methods.55(1):44-51(2011)). in order to determine the optimal concentration range for heterodimer formation, this step was performed with three different ratios of two heavy chain transfected DNA. For example, with 2ml culture volume and transfected DNA consisting of 5% GFP, 45% salmon sperm DNA, 25% light chain and 25% total weight, wherein the heavy chain A plasmid (with C-terminal His tag) and heavy chain B plasmid (with C-terminal StretTagII plus RFP) were sampled at 3 different relative ratios (chain_A (His)/chain_B (mRFP)) of 10%/65%, 20%/55%, 40%/35% or 10%/20%/40%) (determination of apparent 1:1 expression ratio of WT_His/WT_RFP heterodimer was close to DNA ratio 20%/55%). At 4 to 48 hours post-transfection, TN1 peptone was added to a final concentration of 0.5% in F17 serum-free medium (Gibco). The expressed antibodies were analyzed by SDS-PAGE to determine the optimal ratio of heavy and light chains for optimal heterodimer formation (see fig. 25B and C).

The DNA ratios selected as described above, e.g.50% light chain plasmid, 25% heavy chain A plasmid, 25% heavy chain B, with 5% GFP and 45% salmon sperm DNA for AZ33 and AZ34, were used to transfect 150mL cell cultures. Transfected cells were harvested after 5-6 days, centrifuged at 4000rpm, and the culture broth was collected and clarified with a 0.45 μm filter. For each antibody with CH3 mutation generated for further analysis, a list of percentages for light and heavy chain a and B plasmids used in the scaled-up transfection assay, including purity and melting temperature measurements, see table 2 below.

Table 2:

Example 2: purification of bivalent monospecific antibodies with heterodimeric Fc domains.

The clarified broth was loaded onto a MabSelect SuRe (GE HEALTHCARE) protein a column and washed with 10 column volumes of PBS buffer pH 7.2. The antibody was eluted with 10 column volumes of citrate buffer pH 3.6 and the pooled fractions contained antibody neutralized with TRIS at pH 11. Finally, the proteins were desalted using an Econo-Pac 10DG column (Bio-Rad). The C-terminal mRFP tag on heavy chain B was removed by incubating the antibody with enterokinase (NEB) overnight in PBS at a ratio of 1:10,000 at 25 ℃. Antibodies were purified from the mixture by gel filtration. For gel filtration, 3.5mg of antibody mixture was concentrated to 1.5mL and loaded via AKTA Express FPLC to a Sephadex 200 HiLoad 16/600 200pg column (GE HEALTHCARE) at a flow rate of 1 mL/min. PBS buffer at pH 7.4 was used at a flow rate of 1 mL/min. Fractions corresponding to purified antibodies were collected, concentrated to about 1mg/mL and stored at-80 ℃.

The formation of heterodimers compared to homodimers was determined using non-reducing SDS-PAGE and mass spectrometry. Protein a purified antibodies were run on 4-12% gradient SDS-PAGE, non-reducing gels, prior to Enterokinase (EK) treatment to determine the percentage of heterodimers formed (see fig. 26). For mass spectrometry, all Trap LC/MS (ESI-TOF) experiments were performed on an Agilent 1100 HPLC system connected to a Waters Q-TOF2 mass spectrometer. Mu.g of gel-filtered purified antibody was injected into protein MicroTrap (1.0X18.0 mm), washed with 1% acetonitrile for 8 min, gradient of 1 to 20% acetonitrile/0.1% formic acid for 2 min, and then eluted with a gradient of 20 to 60% acetonitrile/0.1% formic acid for 20 min. The eluate (30-50. Mu.L/min) was directed to a spectrometer, and spectra were obtained per second (m/z 800 to 4,000). (see FIG. 28). In addition to AZ12 and AZ14, each of which had greater than 85% heterodimer formation, variants with greater than 90% heterodimer were selected for further analysis.

Example 3: differential Scanning Calorimetry (DSC) was used to determine the stability of bivalent monospecific antibodies with heterodimeric Fc domains.

All DSC experiments were performed using a GE VP-capillary instrument. The protein was buffer exchanged into PBS (pH 7.4) and diluted to 0.4 to 0.5mg/m, 0.137ml was loaded into the sample chamber and measured at a scan rate of 1℃per minute from 20 to 100 ℃. The data was analyzed using Origin software (GE HEALTHCARE) to subtract the PBS buffer background. (see FIG. 27). See table 3 for a list of variations tested and melting temperatures determined. See table 4 for a list of variants having melting temperatures of 70 ℃ and above and specific Tm for each variant.

Table 3: melting temperature measurement of variant CH3 domains in IgG1 antibodies with 90% or more heterodimer formation compared to homodimer formation

Table 4: melting temperature measurement of selected "variant" CH3 domains in IgG1 antibodies

Variants	Tm℃	Variants	Tm℃	Variants	Tm℃	Variants	Tm℃
								Wild type	81.5	AZ42	70	AZ73	71	AZ91	71.5
Control 1	69	AZ44	71.5	AZ74	71	AZ92	71.5
								Control 2	69	AZ46	70.5	AZ75	70	AZ93	71.5
AZ12	＞77	AZ47	70.5	AZ76	71.5	AZ94	73.5
								AZ14	＞77	AZ48	70.5	AZ77	71	AZ95	72
AZ15	71.5	AZ49	71	AZ78	70	AZ98	70
								AZ17	71	AZ63	71.5	AZ79	70	AZ100	71.5
AZ19	70.5	AZ64	74	AZ81	70.5	AZ101	74
								AZ20	70	AZ65	73	AZ82	71	AZ106	74
AZ21	70	AZ66	72.5	AZ83	71	AZ114	71
								AZ25	70.5	AZ67	72	AZ84	71.5	AZ115	70
AZ29	70	AZ68	72	AZ85	71.5	AZ122	71
								AZ30	71	AZ69	71	AZ86	72.5	AZ123	70
AZ32	71.5	AZ70	75.5	AZ87	71	AZ124	70
								AZ33	74	AZ71	71	AZ88	72	AZ129	70.5
AZ34	73.5	AZ72	70.5	AZ89	72.5	AZ130	71

Example 4: assessment of fcγr binding using surface plasmon resonance

All binding experiments were performed with 10mM HEPES,150mM NaCl,3.4mM EDTA, and 0.05%Tween 20 pH 7.4 at 25 ℃ using BioRad ProteOn XPR instrument. Recombinant HER-2/neu (p 185, erbB-2 (eBiosciences, inc.)) was captured on an activated GLM sensing chip by injecting 4.0 μg/mL into 10mM NaOAc (pH 4.5) at 25 μl/min until about 3000 Resonance Units (RU) were immobilized, quenching the remaining active groups. 40 μg/mL of purified anti-Her-2/neu antibody comprising the modified CH3 domain was indirectly captured on the sensor chip by binding Her-2/neu protein when injected at 25 μl/min for 240s (resulting in about 500 RU) after buffer injection to establish a stable baseline. Fcγr (CD 16a (f allotype) and CD32 b) concentrations (6000, 2000, 667, 222, and 74.0 nM) were injected at 60 μl/min for 120s, and 180s dissociation phases to obtain a set of binding sensorgrams. The obtained K _D values were determined from the binding isotherms using a balanced fit model, and the reported values were taken as the average of three independent runs. Comparison was performed with wild-type IgG1 Fc domain and binding was expressed as the ratio of WT kD to variant kD (see, table 5).

Table 5: kD ratio of binding of wild-type IgG1 independent of CD16a and CD32b to modified CH3 domain antibody

/>

Example 5: engineering with Fc_CH3-scaffold 1 (1 a and 1 b) rational design of Fc variants and development of AZ17-62 and AZ133-AZ2438

In order to obtain AZ variants with high stability and purity, the above-described structure and calculation strategies are employed. (see FIG. 24). For example, in-depth structure-function analysis of AZ8 provided a detailed understanding of each introduced mutation of AZ8, l351y_v397 s_f397 a_y407V/k392 v_t394W compared to wild-type human IgG1, and indicated that the important core heterodimer mutation was l351y_f405a_y407V/T394W, whereas V397S, K392V was irrelevant for heterodimer formation. The core mutation (L351 Y_F405 A_Y407V/T394W) is referred to herein as the "scaffold 1" mutation. Furthermore, analysis showed that important interface hot spots for loss of formation of homodimers relative to Wild Type (WT) were the interactions of WT-F405-K409, Y407-T366 and the packaging of Y407-Y407 and-F405 (see FIG. 29). This is reflected in the packaging, cavity and MD analysis, which shows a large conformational difference in the loop D399-S400-D401 (see fig. 30) and the associated β -sheet of K370. This resulted in loss of the interchain interactions of K409-D399 (see fig. 30) and attenuation of the strong hydrogen bonds of K370 and E357 (K370 was no longer in direct contact with S364 and E357, but was completely solvent exposed). In the WT IgG1 CH3 domain, these regions tether the interface at the edges, protecting the core interactions from massive solvent competition, and increasing the dynamic occurrence of favorable hydrophobic van der waals interactions. The result is a lower buried surface area of AZ8 and a higher solvent accessibility of the hydrophobic core compared to WT. This suggests that the most important factors for the lower stability of AZ8 compared to WT stability are a) loss of WT-F405-K409 interactions and packaging of F405, and b) loss of strong packaging interactions of Y407-Y407 and Y407-T366. See, fig. 29.

Thus, we identified a key residue/sequence motif responsible for the low stability of AZ8 compared to WT. In order to improve the stability and heterodimer specificity of AZ8, subsequent positive design engineering efforts were therefore focused on stabilizing the loop conformation at positions 399-401 in a more 'closed' -WT like conformation (see figure 30) and compensating for the slightly reduced overall (more relaxed) packaging of the hydrophobic core at positions T366 and L368 (see figure 29).

To achieve this stability of the loop conformation at positions 399-401, the computational method was used to evaluate different targeting design ideas. In particular, 3 different independent selections for Fc variant AZ8 were analyzed to optimize the identified critical regions for improved stability. First, the better hydrophobic packaging of the binding pocket near positions K409 and F405A was evaluated in order to both protect the hydrophobic core and stabilize the ring conformation of 399-400 (see figure 30). These include, but are not limited to, additional point mutations at positions F405 and K392. Next, the selection for improving electrostatic interactions at positions 399-409 was evaluated to stabilize the ring conformation of 399-400 and to protect the hydrophobic core. This includes, but is not limited to, additional point mutations at positions T411 and S400. Third, the binding pockets at the T366, T394W and L368 core packaging positions were evaluated to improve the core hydrophobic packaging (see fig. 29). These include, but are not limited to, additional point mutations at positions T366 and L368. Different independent positive design ideas were tested on a computer chip and experiments validated certain good variants (AZ 17-AZ 62) using a computational tool for expression and stability as described in examples 1-4. For a list of certain Fc-based heterodimer constructs with melting temperatures of 70 ℃ or higher that included this design strategy, see table 4.

Fc variant AZ33 is an example of the development of Fc variants in which scaffold 1 was modified resulting in mutation of scaffold 1a to improve stability and purity. The Fc variant was designed based on AZ8 with the aim of improving the hydrophobic packaging at positions 392-394-409 and 366 in order to both protect the hydrophobic core and stabilize the loop conformation of 399-400. The Fc variant AZ33 heterodimer has two additional point mutations different from the core mutations AZ8, K392M and T366I. The mutation T366i_k392m_t394W/f405a_y407V is referred to herein as the "scaffold 1a" mutation. The mutation K392M was designed to improve the packaging of the cavities near positions K409 and F405A in order to protect the hydrophobic core and stabilize the ring conformation of 399-400 (see fig. 31). T366I was designed to improve core hydrophobic packaging and eliminate the formation of homodimers of the T394W chain (see figure 29). Experimental data for AZ33 showed significantly improved stability relative to other negative design Fc variants such as AZ8 (Tm 68 ℃) where AZ33 had a Tm of 74 ℃ and a heterodimer content of > 98%. (see, fig. 25C).

Development of Fc variants Using scaffold 1 mutations in the third stage design of Fc variant heterodimers

Although AZ33 provides significant stability and specificity (or purity) improvements over the initial starting variant AZ8, our analysis shows that further improvements in stability of Fc variant heterodimers can be made with further amino acid modifications and using experimental data for AZ33 and the design methods described above. The expression and stability of the different design concepts have been tested independently, but the independent design concepts are transferable and the most successful heterodimers will contain a combination of different designs. In particular, to optimize AZ8 packaging, mutations at cavities near K409-F405A-K392 have been evaluated independent of optimizing the mutation of the core packaging at residue L366T-L368. The two regions 366-368 and 409-405-392 are remote from each other and are considered to be independent. For example, the Fc variant AZ33 has been optimized with respect to packaging at 409-405-392, but not at 366-368, as these optimized mutations were evaluated separately. Comparison of the 366-368 mutations shows that T366L has improved stability compared to T366 and also T366I (point mutation used in the development of Fc variant AZ 33). Thus, the experimental data presented immediately demonstrate further optimisation of AZ33 by the introduction of T366L instead of T366I (for example). Thus, the amino acid mutation in the CH3 domain, T366l_k392m_t394W/f405a_y407V, is referred to herein as a "scaffold 1b" mutation.

In a similar manner, complete experimental data have been analyzed to identify point mutations that can be used to further improve the current Fc variant heterodimer AZ 33. These identified mutations were analyzed by the calculation method described above and ordered to generate a list of additional Fc variant heterodimers based on AZ33, as shown in table 6.

Example 6: rational design of Fc variants and development of AZ63-101 and AZ2199-AZ2524 Using Fc-CH 3 engineering-scaffold 2 (a and b)

To improve the stability and purity of the Fc variant AZ15 at the initial negative design stage, the above structure and calculation strategy was employed (see fig. 24). For example, in-depth structure-function analysis of Fc variant AZ15 provided a detailed understanding of each of the introduced mutations of AZ15, l351y_y407A/E357l_t366a_k409f_t411N compared to wild-type (WT) human IgG1, and indicated that the important core heterodimer mutation was l351y_y407A/T366a_k409F, whereas E357L, T411N was not directly related to heterodimer formation and stability. The core mutation (L351 Y_Y407A/T366 A_K409F) is referred to herein as the "scaffold 2" mutation. Furthermore, analysis showed that important interface hot spots lost to Wild Type (WT) homodimer formation were salt bridges D399-K409, hydrogen bonds Y407-T366, and packaging Y407-Y407. The detailed analysis provided below describes how we improve our initial Fc variant AZ15 and the position and amino acid modifications made to the Fc variants that achieve these improved stabilities.

The scaffold 2 mutations were used to develop Fc variants and further to develop scaffold 2a mutations.

Computer on-chip analysis showed that previous Fc variants designed non-optimal packaging of e.g. AZ15 mutation k457fjt366A jy407A and overall reduced packaging of hydrophobic cores due to loss of WT-Y407 interactions. The heteromultimers described herein are designed with a more optimal packaging. Some of the positive design efforts described herein focused on point mutations to compensate for packaging defects in the initial Fc variant AZ 15. Targeting residues include T366, L351, and Y407. Different combinations of these were tested on a computer chip and the best ranked Fc variant (AZ 63-AZ 70) using a computational tool was verified with respect to expression and stability experiments, as described in examples 1-4.

Fc variant AZ70 is an example of the development of Fc variants in which scaffold 2 was modified resulting in mutations in scaffold 2a to improve stability and purity. The Fc variant was designed based on AZ15 with the aim of achieving better packaging of the hydrophobic core as described above. Fc variant AZ70 has the same scaffold 2 core mutation (l351 y_y407A/T366 a_k409F) as described above, except that T366 was mutated to T366V instead of T366A (fig. 33). The L351Y mutation improved the melting temperature of the 366a_409f/407A variant from 71.5 ℃ to 74 ℃, and the additional change from 366A to 366V improved the Tm to 75.5 ℃. (see, AZ63, AZ64 and AZ70 in Table 4, which have Tm of 71.5 ℃, 74 ℃ and 75.5 ℃ respectively). The core mutation (L351 Y_Y407A/T366 V_K409F) is referred to herein as the "scaffold 2a" mutation. Experimental data for the Fc variant AZ70 showed significantly improved stability compared to the initial negative design Fc variant AZ15 (Tm 71 ℃), where AZ70 had a Tm of 75.5 ℃ and a heterodimer content of >90% (fig. 33 and 27).

The scaffold 2 mutations were used to develop Fc variants and further to develop scaffold 2b mutations.

Molecular dynamics simulation (Molecular Dynamics simulation, MD) and packaging analysis showed the preferred more 'open' conformation of loops 399-400, possibly due to loss of WT salt bridge K409-D399. This also results in unsatisfactory D399, which in turn prefers compensatory interactions with K392 and induces a more 'open' conformation of the loop. This more 'open' loop conformation results in overall reduced packaging and higher solvent accessibility of the core CH3 domain interface residues, which in turn significantly reduces the stability of the heterodimer. Thus, one of the targeted positive design efforts was to tether the loop in a more 'blocked' WT-like conformation by compensating for additional point mutations in the loss of packaging interactions of the D399-K409 salt bridge and K409. Targeting residues include T411, D399, S400, F405, N390, K392, and combinations thereof. Different packaging, hydrophobicity and electrostatic positive engineering strategies were tested on a computer chip for the above positions and the best ranked Fc variants (AZ 71-AZ 101) determined using a computational tool were verified with respect to expression and stability experiments, as described in examples 1-4.

Fc variant AZ94 is an example of the development of Fc variants in which scaffold 2 was modified resulting in scaffold 2b mutations along with additional point mutations to improve stability and purity. The Fc variants were designed based on the objective of tethering loops 399-400 in a more 'blocked' WT-like conformation and compensating for the loss of D399-K409 salt bridge as described above. The Fc variant AZ94 had 4 additional point mutations to scaffold 2 (L351 y_y407A/T366 a_k409F) and L351Y was reverted to wild-type L351, leaving (Y407A/T366 a_k409F) as the core mutation for the Fc variant. The core mutation Y407A/T366A_K409F is referred to herein as the "scaffold 2b" mutation. Four additional point mutations of AZ94 are K392E_T411E/D399R_S400R. The mutation T411E/D399R was engineered to form additional salt bridges and offset the loss of K409/D399 interaction (FIG. 34). In addition, such salt bridges are designed to prevent homodimer formation by excluding charge-charge interactions in the two potential homodimers. The additional mutation K392E/S400R was intended to form another salt bridge, and thus further tethered the 399_400 loop in a more "blocked" WT-like conformation (fig. 34). Experimental data for AZ94 showed improved stability and purity relative to the original negative design Fc variant AZ15 (Tm 71 ℃, >90% purity), wherein Fc variant AZ94 has Tm 74 ℃ and a heterodimer content or purity of > 95%.

Development of Fc variants using scaffold 2 mutations in the third stage design of Fc variant heterodimers

Fc variants AZ70 and AZ94 provided significant improvements in stability and purity over the initial negative design Fc variants such as AZ15, but our analysis and comparison of AZ70 and AZ94 directly indicated that further amino acid modifications could be used to make improvements in the expectation of stability of Fc variant heterodimers. For example, fc variants AZ70 and AZ94 were designed to target two different non-optimal regions in the initial variant AZ15 by improving the packing of the hydrophobic core and mutating outside the core interface residues, resulting in additional salt bridges and hydrogen bonding to stabilize the loop conformation at positions 399-401. The additional point mutations of Fc variants AZ70 and AZ94 are distant from each other and are therefore independent and transferable to other Fc variants designed around the same scaffold 2 core mutation (including the 2a and 2b mutations). Specifically, AZ70 carries only the optimized core mutation l351 y_y407A/T366 a_k409F, but no additional salt bridge, while AZ94 contains four additional electrostatic mutations (k392 e_t411E/D399 r_s400R), but one less mutation in the hydrophobic core interface (Y407A/T366 a_k409F). These scaffold 2b mutations are less stable than AZ70 (see, e.g., AZ63, which has an equivalent core mutation to AZ94 and a Tm of 72 ℃) but are compensated for by the addition of the k392e_t411E/D399r_s400r mutation. The experimental stability and purity data provided indicate that mutations that bind to AZ70 (which optimizes the hydrophobic core and electrostatic mutation of AZ 94) should further improve the stability and purity of Fc variant heterodimers. In a similar manner, the complete experimental data for the scaffold 2Fc variant (AZ 63-101) have been analyzed to identify point mutations that can be used to further improve Fc variant heterodimers AZ70 and AZ 94. These identified mutations were further analyzed by the calculation method described above and ordered to generate a list of additional Fc variant heterodimers based on AZ70 and AZ94, as shown in table 7.

Example 7: effect of heterodimer CH3 on FcgR binding

As typical examples of the activity of heterodimeric Fc with FcgR, two variant antibodies A: K459D_K392D/B: D399K_D356K (control 1 (het 1) in FIG. 35) and A: Y346C_T366 S_L368A_Y407V/B: S354C_T366W (control 4 (het 2) in FIG. 35) having a heterodimeric Fc region with a Her2 binding Fab arm have been tested in an SPR assay as described for FcgR binding in example 4. As shown in fig. 35, we observed that both heterodimeric Fc regions bound different fcγ receptors with the same relative intensity as the wild-type IgG1 Fc region, but overall, the heterodimeric Fc regions bound slightly better to each FcgR than the wild-type antibody. This suggests that mutations at the CH3 interface of Fc can affect the binding strength of the Fc region across the CH2 domain to fcγ receptors, as observed in molecular dynamics simulations and analysis.

Example 8: effect of asymmetric mutations in CH2 of heterodimeric Fc on FcgR binding

The serine mutation in the CH2 domain of the Fc region at position 267 to aspartic acid (S267D) is known to enhance binding to fcγiibf, IIbY & IIaR receptors when introduced in a homodimeric manner into both chains of the CH2 domain. Such mutations can be introduced into only one CH2 domain of the heterodimeric Fc molecule to obtain an improvement in binding strength relative to about half when such mutations are introduced into the homodimeric CH2Fc, as demonstrated by the data provided in fig. 36A. On the other hand, the E269K mutation in the CH2 domain of the homodimer of Fc prevents binding of the Fc region to FcgR. We propose a solution to enhance the binding strength of the operating Fc region to Fcg receptors by asymmetrically introducing these favorable and unfavorable mutations on one of the two chains in the CH2 domain of Fc. Introduction of the E269K mutation in an asymmetric manner onto one CH2 chain of the heterodimer Fc acts as a polar driver by blocking FcgR binding at the face where it exists while allowing the other face of the Fc to interact with FcgR in the normal manner. The results from these experiments are provided in fig. 36A. The opportunity to selectively alter binding strength via both chains of the Fc in an independent manner provides an opportunity to manipulate the increase in binding strength and selectivity between Fc and Fcg receptors. Thus, this asymmetric design of mutations in the CH2 domain enables us to introduce positive and negative design strategies to support or not support certain binding models, providing more opportunities for introducing selectivity.

In subsequent experiments, we have altered the selectivity profile of the basic Fc mutant s239d_d265s_i332e_s298A, which shows increased binding strength to fcγ IIIaF and IIIaV receptors, while continuing to show weaker binding to fcγiiar, IIbF and IIbY receptors. This is shown in the binding profile shown in fig. 36B. By introducing an asymmetric mutation E269K in chain a and avoiding the I332E mutation in chain B, we were able to generate a new FcgR binding profile that further attenuated IIa and IIb receptor binding and made Fc more specific for IIIa receptor binding.

In another example shown in FIG. 36C, an asymmetric mutation relative to homodimer Fc is highlighted, which involves the mutation S239D/K326E/A330L/I332E/S298A in the CH2 domain. This variant showed increased binding to the IIIa receptor relative to wild-type IgG1 Fc, but also bound slightly stronger to the IIa and IIb receptors than wild-type Fc. The introduction of these mutations in an asymmetric manner A: S239D/K326E/A330L/I332E and B: S298A, while reducing IIIa binding, also increases IIa/IIb receptor binding, in the course of which selectivity is lost. IIa/IIb binding was reduced back to wild-type levels by introducing an asymmetric E269K mutation (i.e., A: S239D/K326E/A330L/I332E/E269K and B: S298A) in this heterodimeric variant. This highlights the fact that: the use of asymmetric mutations in the CH2 domain of Fc can provide an important opportunity to design improved fcγr selectivity.

The reagents employed in the examples are commercially available or may be prepared using commercially available instruments, methods, or reagents known in the art. The above examples illustrate various aspects of the invention and the practice of the methods of the invention. The examples are not intended to provide an exhaustive description of the many different embodiments of the invention. Accordingly, while the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art that certain changes and modifications may be made thereto without departing from the spirit and scope of the appended claims.

Example 9: fcRn binding as determined by SPR.

Binding to FcRn was determined by SPR in two different orientations.

1. Flowing the heterodimeric variant through an immobilized FcRn: in this experiment, a high density surface of about 5000RU was prepared using standard NHS/EDC coupling. In triplicate, 100nM WT and each variant were injected at 50uL min for 120s and dissociated in MES pH6 running buffer for 600s.

2. FcRn is passed through indirectly captured heterodimeric variants: in this SPR experiment, a goat anti-human IgG surface was used to indirectly capture antibodies (about 400RU each) followed by injection of a 3-fold FcRn dilution series (6000 nM high concentration). The running buffer was 10mM MES/150mM NaCl/3.4mM EDTA/0.05 Tween20 at pH 6. FcRn did not bind significantly to the goat polyclonal surface. All variants showed similarity to WT sensorgrams. Table 8 below shows Kd as determined by indirect immobilization with streaming FcRn (2.).

Table 8: kd determined by indirect immobilization with streaming FcRn

Example 10: bispecific binding of Fc heterodimers described herein

Use of a mutant chain-a: l351y_f405a_y407V, chain-B: the Fc heterodimer of T366l_k392m_t394W and the anti-HER 2 and anti-HER 3 scFv fused to the N-terminus of chain-a and chain-B of the Fc heterodimer confirmed bispecific binding. The resulting variant bispecific HER2/HER3 variants and two monovalent-monospecific HER2, HER3 variants are illustrated in figure 40A. To test for bispecific binding, a range of doses of two monovalent variants (anti-HER 2 monovalent and anti-HER 2 monovalent, illustrated in fig. 40A) and bispecific anti-HER 2/HER3 heterodimers were incubated with a MALME-M3 melanoma molecule followed by FACS analysis to determine the apparent binding affinity of each molecule (shown in fig. 40B). The assay system ："Antitumor activity of a novel bispecific antibody that targets the ErbB2/ErbB3 oncogenic unit and inhibits heregulin-induced activation of ErbB3", McDonagh CF et al, mol Cancer Ther.11 (3): 582-93 (2012) was set up according to the protocol described in the following document.

Example 11: expression and purification of bivalent monospecific antibodies with heterodimeric Fc domains and quantification of purity by LC/MS

Heterodimeric variants AZ133(A： L351Y/F405A/Y407V,B：T366L/K392M/T394W)、AZ138(A： F405A/Y407V,B：T366L/K392M/T394W)、AZ3002(A：T350V/L351Y/F405A/Y407V,B：T350V/T366L/K392M/T394W)、 AZ3003(A：T350V/L351Y/F405A/Y407V,B： T350V/T366L/K392L/T394W) and other AZ constructs AZ3000-AZ3021 were generated and purified as described in examples 1 and 2. To assess the robustness of heterodimer formation and the effect of excess one heterodimer chain on heterodimer purity, 3 different DNA ratios of the two heavy chains a and B (e.g., ratio a: b=1:1.5; 1:1; 1.5:1) were used to transiently express the selected heterodimer.

Genes encoding heterodimeric heavy and light chains were constructed via gene synthesis using codons optimized for human/mammalian expression as detailed in example 1. Fab sequences were generated from known Her2/neu binding abs (Carter p. Et al (1992)Humanization of an anti P185 Her2 antibody for human cancer therapy.Proc Natl Acad Sci 89,4285) and Fc is IgG1 isotype (SEQ ID NO: 1). As described in examples 1-2, variants were expressed by transient co-expression using 3 different heavy chain-a and heavy chain-B ratios of 1:1.5, 1:1 and 1.5:1. Purified samples were purified by protein-a affinity chromatography and preparative gel filtration (see details of example 2). The purified samples were deglycosylated overnight with PNGaseF at 37 ℃, samples were injected into Poros R2 columns and eluted with a gradient of 20-90% acn, 0.2% fa for 3 minutes prior to MS analysis. Peaks of LC columns were analyzed with LTQ-Orbitrap XL mass spectrometer (cone aperture voltage: 50V' column: 215V ft resolution: 7,500) and integrated with software Promass to generate molecular weight characteristics.

The relative peak heights of the heterodimers and homodimers were used to evaluate heterodimer purity (see fig. 39).

Example 12: crystal structures of heterodimers AZ3002 and AZ 3003:

Heterodimeric Fc constructs of AZ3002 and AZ3003 were expressed in CHO and purified to homogeneity by pA and SEC. After incubation at a ratio of 2:1 for about 24 hours via hanging-drop vapor diffusion method with the aid of micro-inoculation in the above-mentioned mother liquor solution consisting of 5% (v/v) ethylene glycol, 18% (w/v) polyethylene glycol 3350 and 0.15M ammonium iodide, purified Fc heterodimer was crystallized at 18 ℃. The crystals were cryoprotected by increasing the ethylene glycol concentration to 30% (v/v) and then flash cooling in liquid nitrogen. Diffraction data for both crystals were collected at 100K using 0.5 degree amplitude for a total of 200 degrees and treated ¹ with XDS. Using PDBID:2J6E was used as a query protein to resolve structure ² of AZ3002 via molecular substitution using Phaser. AZ3003 was then resolved in a similar manner using the structure of AZ 3002. To provide perfect bimorphs, the reciprocal relationship of asymmetric heterodimers (two possible heterodimer pairs each with an atomic occupancy of 0.5) present in asymmetric units of crystallography (e.g., the occupancy of molecule a can be similarly described by molecule B and vice versa) is modeled ^3,4 with Refmac-modified Coot. Diffraction data processing and structure correction statistics are shown in table 9.

Table 9:

1.Kabsch，W.XDS.Acta Crystallogr D Biol Crystallogr 66，125-132(2010).

2.McCoy,A.J.Solving structures of protein complexes by molecular replacement with Phaser.Acta Crystallogr.D.Biol.Crystallogr.63,32-41(2007).

3.Emsley,P.&Cowtan,K.Coot：model-building tools for molecular graphics.Acta Crystallogr.D.Biol.Crystallogr.60,2126-2132(2004).

4.Murshudov,G.N.,Vagin,A.A.&Dodson,E.J.Refinement of macromolecular structures by the maximum-likelihood method.Acta Crystallogr.D.Biol.Crystallogr.53,240-255(1997).

the overlay of the crystal structure is shown in fig. 42. The crystal structures of AZ3002 and AZ3003 heterodimers showed very good agreement with the on-chip model of the computer ) And conforms to the predicted conformation of the critical core packaging residues.

Example 13: glycosylation analysis of AZ3003

AZ3003 heterodimer was expressed and purified as described in example 11. Glycans were analyzed using GlykoPrep ^TM Rapid N-Glycan Preparation and InstantAB ^TM (Prozyme) using standard manufacturer protocols.

The results are shown in fig. 43 and illustrate that AZ3003 has a typical glycosylation pattern.

Example 14: evaluation of stability of AZ3003 under forced degradation conditions

The stability of AZ3003 heterodimers was assessed by incubation under forced degradation conditions. Stability of mabs under forced degradation conditions can be a good assessment for long term and formulation stability.

Purified heterodimer samples (expressed and purified as described in example 11) were concentrated to 100mg/ml without signs of aggregation. The samples were diluted into suitable buffers and evaluated under forced degradation conditions as described in table 10 below. The treated samples were analyzed by SDS-PAGE and HPLC-SEC.

SDS-PAGE was performed with pre-prepared gradient gels from LONZA under reducing (R) and non-reducing (NR) conditions. Protein bands were visualized by staining with Coomassie Brilliant Blue G-250.

Analytical SEC-HPLC was performed using Phenomenex, BIOSEP-SEC-S4000 or BioRad Bio-Sil TSK 4000 HPLC column with 10mM sodium phosphate, 0.14M NaCl, 10% isopropanol as running buffer at a flow rate of 0.8 ml/min. This allows for quantification of potentially higher and lower molecules and species.

Table 10: forced degradation conditions for degrading AZ3003

The results are shown in figure 44 and demonstrate that AZ3003 heterodimer is stable and exhibits stability characteristics consistent with the stability of industry standard mabs.

Example 15: downstream purification evaluation of AZ3003

Manufacturability evaluation of AZ3003 was performed to evaluate the behavior of AZ3003 using the industry standard antibody purification method flow as shown in fig. 45. The method involves a three column step platform that includes protein a affinity chromatography for product capture followed by Cation Exchange (CEX) chromatography for aggregate removal, leached protein a and HCP, and finally Anion Exchange (AEX) chromatography in flow-through mode to capture viruses, DNA and negatively charged contaminants. This assessment is used to identify potential manufacturing problems (e.g., process stability, product stability, and quality) of the drug candidate early in the research/development stage.

During manufacturability evaluation, chromatographic behavior, protein stability and product quality were evaluated using the industry standard purification method shown in fig. 46. Table 11 (below) lists the main criteria used to evaluate i.e. step yield, high molecular weight aggregate (HMW) content and elution volume. High-step yields and low-elution volumes during purification indicate a well-functioning, stable protein. Monitoring of the HMW content and its removal during purification is critical, as the presence of protein aggregates (HMW species) in the final product can lead to a reduction of the active immunogenic response in the patient and/or microparticle formation during the half-life of the drug product. Expression of mabs with minimal initial HMW content (< 4%) is desirable because high levels of aggregates would require additional purification steps for removal, thereby increasing time and cost of manufacture.

Table 11: main criteria for manufacturability evaluation

The stability, chromatographic behavior and product quality of AZ3003 were verified using standard industrial purification methods.

1.1 Protein A Capture

CM expressing AZ3003 was 0.22- μm filtered using a bottle cap filter (PES) from Millipore and applied to a Mab Select SuRe (1.6x25 CM) column equilibrated with 5CV of 20mM Tris-HCl, 0.14M NaCl, pH 7.5. After loading, the column was thoroughly washed with equilibration buffer until the a280 absorbance reached a stable baseline. AZ3003 was eluted with 0.1M acetate buffer (pH 3.6) and immediately titrated to pH 5.2 by adding 1/10 of the volume of 1M tris base.

After the elution step, the column was washed with 0.1M acetate (pH 3.0). SDS-PAGE analysis showed that all the mabs bound to the column, since no Mab was detected in column FT. Highly purified Mab was detected in pH 3.6 elution buffer. The initial capture and purification steps using protein a affinity chromatography gave a product with a purity > 90%.

1.2 Low pH maintenance study

The next step in the downstream process is a low pH hold, which is performed in order to inactivate the virus. After elution from the protein a column, mab (about 10mg/ml, pH 4.0) was titrated to pH 3.6 with 10% acetic acid and incubated for 90min at RT with stirring. Stability of AZ3003 against low pH treatment was assessed by SDS-PAGE, SEC-HPLC and by turbidity measurement at a410 nm. AZ3003 was well tolerated for the low pH maintenance step and showed no change in SDS-PAGE or SEC-HPLC. In addition, an increase in turbidity was detected after 90-min incubation, indicating the lack of formation of insoluble aggregates, which can be a troublesome problem during purification (i.e., clogging filters and columns in the process, loss of product). These data show that AZ30003 is stable to low pH maintenance procedures.

1.3 Cation exchange Chromatography (CEX)

CEX was studied as a second step in the purification process. The following two resins were evaluated: fractogel EMD SO3 (M) from Merk/Millipore and SP HP from GE LIFESCIENCES.

Fractogel EMD SO3 (M), pH 5.2: the Mab Select SuRe mixture (35 mg) was titrated to pH 5.2 by the addition of 10% (v/v) 1M tris base and then 2-fold diluted with equilibration buffer 20mM acetate (pH 5.2). The mixture was applied to a Fractogel EMD SO3 (M) column equilibrated with 5CV of 20mM acetate (pH 5.2). The column was washed with equilibration buffer until the a280 absorbance reached a stable baseline. The Mab was eluted from the column with a linear salt gradient of 0 to 600mM NaCl (pH 5.2) over 10 CV. The remaining contaminants were stripped from the column with 20mM acetate, 1M NaCl, pH 5.2, followed by treatment with 1N NaOH. SDS-PAGE and SEC-HPLC analysis was performed to monitor HMW levels and their removal from the main Mab fraction of the column. The step yield (based on A280nm reading) was 73%.

SP HP, pH 5.2: the Mab Select SuRe mixture (50 mg) was titrated to pH 5.2 by the addition of 10% (v/v) 1M tris base and then diluted likewise with equilibration buffer 20mM acetate (pH 5.2). The mixture was applied to an SP HP column (1.6X2.5 cm/5 ml) equilibrated with 5CV of 20mM acetate (pH 5.2) (FIG. 21). The column was washed with equilibration buffer until the a280 absorbance reached a stable baseline. The Mab was eluted from the column with a linear salt gradient of 0 to 600mM NaCl (pH 5.2) over 10 CV. The remaining contaminants were stripped from the column with 20mM acetate, 1M NaCl, pH 5.2, followed by treatment with 1N NaOH. SDS-PAGE and SEC-HPLC analysis was performed to monitor the level of aggregation and its separation on the column.

The step yield (based on A280nm reading) was 87%.

1.4 Anion exchange chromatography (AEX)

Anion exchange (e.g., quaternary amine, Q) has been widely used for monoclonal antibody purification. AEX medium was operated in flow-through mode, with Mab present in FT while allowing retention of HCP, DNA, virus and endotoxin.

The Fractogel SO 3M mixture pH 5.2 (25 mg) was titrated with 1M tris base to pH 7.0 and applied to 1ml HiTrap Q FF equilibrated with 5CV of 10mM phosphate (pH 7.0). The column was washed with equilibration buffer. In this case, however, a stable baseline is not reached. Thus, the column was washed with PBS to elute any residual bound Mab. The column was then washed with 10mM phosphate, 1M NaCl, pH 7.0 to remove any bound contaminants. This step requires further optimization to have all Mab fractions present in the flow-through mode. The step yield (based on A280nm reading) was 82% and purity >98% was estimated by SEC-HPLC.

1.5 SDS-PAGE analysis of downstream purification

The eluate from each purification step was subjected to SDS-PAGE (fig. 35) to monitor contaminant removal and to evaluate the final product quality and purity throughout the process. Gel analysis showed migration patterns of the desired Mab under non-reducing and reducing conditions. Gel comparison from a mixture of two CEX resins showed no major differences in product properties. The main mixtures from CEX, CHT and HIC pH 5.0 and pH 7.0 all appeared similar for purity and contaminating bands.

1.7 Evaluation of purity by SEC-HPLC

Protein a was evaluated by SEC (size exclusion) -HPLC under native conditions in a three column chromatography step; CEX; AEX flow-through mode followed by purified AZ3003 (fig. 46).

AZ3003 showed a single peak eluting within the 150kDa region of the desired native IgG 1. Purity was estimated to be >98%.

1.8 Process yield for purification of AZ3003

The process yields for the downstream purification processes were calculated and are listed in figure 46. Step yields of AZ3003 are typical for IgG purified using industry standard three column purification methods.

AZ3003 was successfully purified from CM using industry standard purification methods (protein a affinity resin, CEX followed by AEX).

These results indicate that AZ3003 was similar to the standard mAb in overall recovery yield (see keley b. Biotechnol. Prog.2007,23, 995-1008) and aggregation was minimally observed. AZ3003 was also evaluated for low pH hold & CHT (ceramic hydroxyapatite) and HIC (hydrophobic interaction chromatography) (PHENYL HP PH and pH 7), with good recovery without aggregates. FIG. 46 illustrates that the lead heterodimer was successfully purified from CM using industry standard purification methods (protein A affinity resin, CEX followed by AEX).

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

Claims

1. An isolated heteromultimeric Fc construct comprising a modified heterodimeric CH3 domain compared to a wild-type CH3 domain, the modified heterodimeric CH3 domain comprising:

A first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, wherein amino acid substitutions in each of the first and second modified CH3 domain polypeptides promote formation of a heterodimeric CH3 domain as compared to a homodimeric CH3 domain; wherein the method comprises the steps of

(A) Amino acid substitutions in the first modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain consisting of amino acid substitutions T350V, F405A, Y407V, and optionally S400R, amino acid substitutions in the second modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain consisting of amino acid substitutions T350V, T366L, K392M, T394W, and optionally N390D or N390E, or

(B) Amino acid substitutions in the first modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain consisting of amino acid substitutions T350V, amino acid substitutions F405A, amino acid substitutions Y407V, amino acid substitutions S400E, and optionally amino acid substitutions Q347R, and amino acid substitutions in the second modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain consisting of amino acid substitutions T350V, amino acid substitutions T366L, amino acid substitutions K392M, amino acid substitutions T394W, amino acid substitutions N390R, and optionally amino acid substitutions K360E, or

(C) The amino acid that promotes heterodimerization of the CH3 domain formed in the first modified CH3 domain polypeptide consists of amino acid substitution T350V, amino acid substitution L351Y, amino acid substitution F405A, F S, F T or F405V, amino acid substitution Y407V, and optionally amino acid substitution S400R, and the amino acid substitution that promotes heterodimerization of the CH3 domain formed in the second modified CH3 domain polypeptide consists of amino acid substitution T350V, amino acid substitution T366L, amino acid substitution K392L or K392M, amino acid substitution T394W, and optionally amino acid substitution N390D or N390E, or

(D) An amino acid substitution in the first modified CH3 domain polypeptide that promotes formation of a heterodimeric CH3 domain consisting of amino acid substitution T350V, amino acid substitution L351Y, amino acid substitution F405A, F405S, F T or F405V, amino acid substitution Y407V, amino acid substitution S400E, and optionally amino acid substitution Q347R, and an amino acid substitution in the second modified CH3 domain polypeptide that promotes formation of a heterodimeric CH3 domain consisting of amino acid substitution T350V, amino acid substitution T366L, amino acid substitution K392L or K392M, amino acid substitution T394W, amino acid substitution N390R, and optionally amino acid substitution K360E;

Wherein the first and second modified CH3 domain polypeptides form a heterodimeric CH3 domain having a melting temperature (Tm) between 74 ℃ and 83 ℃ and a purity of at least 95%;

wherein the heteromultimeric Fc construct is based on human IgG1 and

Wherein the numbering of amino acids is according to the EU index as described in Kabat.

2. The isolated heteromultimeric Fc-construct of claim 1 (c), wherein said amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is comprised of amino acid substitutions T350V, L Y, F a and Y407V, and said amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is comprised of amino acid substitutions T350V, T366L, K392L or K392M, and T394W.

3. The isolated heteromultimeric Fc-construct of claim 1 (c), wherein the amino acid substitutions in the first modified CH3 domain polypeptide that promote heterodimerization in the CH3 domain are composed of amino acid substitutions T350V, L351Y, F a and Y407V, and the amino acid substitutions in the second modified CH3 domain polypeptide that promote heterodimerization in the CH3 domain are composed of amino acid substitutions T350V, T366 37392M and T394W.

4. The isolated heteromultimeric Fc-construct of claim 1 (c), wherein the amino acid substitutions in the first modified CH3 domain polypeptide that promote heterodimerization in the CH3 domain are composed of amino acid substitutions T350V, L351Y, F a and Y407V, and the amino acid substitutions in the second modified CH3 domain polypeptide that promote heterodimerization in the CH3 domain are composed of amino acid substitutions T350V, T366L, K L and T394W.

5. The isolated heteromultimeric Fc-construct of claim 1 (c), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351, Y, S, 400, R, F a and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366, L, K392M and T394W.

6. The isolated heteromultimeric Fc-construct of claim 1 (d), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351Y, S400E, F a and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366L, N390 35392M and T394W.

7. The isolated heteromultimeric Fc-construct of claim 1 (d), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351Y, S400E, F V and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366L, N390 35392M and T394W.

8. The isolated heteromultimeric Fc-construct of claim 1 (d), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351Y, S400E, F T and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366L, N390 35392M and T394W.

9. The isolated heteromultimeric Fc-construct of claim 1 (d), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351Y, S400E, F S and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366L, N390 35392M and T394W.

10. The isolated heteromultimeric Fc-construct of claim 1 (b), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, S400E, F a and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366L, N390R, K392M and T394W.

11. The isolated heteromultimeric Fc-construct of claim 1 (d), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351, Y, S, 400, E, F a and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L, 351, Y, T, 366, L, N, R, K392M and T394W.

12. The isolated heteromultimeric Fc-construct of claim 1 (d), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions Q347R, T350V, L351Y, S400E, F a and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, K360E, T366L, N390R, K392M and T394W.

13. The isolated heteromultimeric Fc-construct of claim 1 (c), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351Y, S400R, F a and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366L, N390 35392M and T394W.

14. The isolated heteromultimeric Fc-construct of claim 1 (c), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351Y, S400R, F a and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366L, N390 35392M and T394W.

15. The isolated heteromultimeric Fc-construct of claim 1 (d), wherein the amino acid substitution in the first modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, L351Y, S400E, F a and Y407V, and the amino acid substitution in the second modified CH3 domain polypeptide that promotes heterodimerization in the CH3 domain is composed of amino acid substitutions T350V, T366L, N390R, K L and T394W.

16. The isolated heteromultimeric Fc-construct of claim 1 (a), wherein the amino acid substitutions in the first modified CH3 domain polypeptide that promote heterodimerization in the CH3 domain are composed of amino acid substitutions T350V, F a and Y407V, and the amino acid substitutions in the second modified CH3 domain polypeptide that promote heterodimerization in the CH3 domain are composed of amino acid substitutions T350V, T366L, K M and T394W.

17. An isolated heteromultimeric Fc-construct comprising a modified heterodimeric CH3 domain compared to a wild-type CH3 domain, said modified heterodimeric CH3 domain comprising a first modified CH3 domain polypeptide and a second modified CH3 domain polypeptide, wherein amino acid substitutions in each of said first and second modified CH3 domain polypeptides promote the formation of a heterodimeric CH3 domain compared to a homodimeric CH3 domain, wherein:

(a) Amino acid substitutions in the first modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain are comprised of amino acid substitutions T350V, L351Y, S400E, F a and Y407V, and amino acid substitutions in the second modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain are comprised of amino acid substitutions T350V, T366L, N390R, K392F and T394W; or (b)

(B) Amino acid substitutions in the first modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain consisting of amino acid substitutions Y349C, T350V, L351Y, S E, F A and Y407V, and amino acid substitutions in the second modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain consisting of amino acid substitutions T350V, S354C, T366L, N R, K392M and T394W, or

(C) The amino acid that promotes heterodimerization of the CH3 domain in the first modified CH3 domain polypeptide consists of amino acid substitutions Y349C, T350V, S400E, F A and Y407V, and the amino acid substitution that promotes heterodimerization of the CH3 domain in the second modified CH3 domain polypeptide consists of amino acid substitutions T350V, S354C, T366L, N390R, K392M and T394W, or

(D) Amino acid substitutions in the first modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain formation consisting of amino acid substitutions Y349C, T350, 350V, F405A and Y407V, and amino acid substitutions in the second modified CH3 domain polypeptide that promote heterodimerization of the CH3 domain formation consisting of amino acid substitutions T350V, S354C, T366L, K392M and T394W;

wherein the heteromultimeric Fc construct is based on human IgG1 and

18. The isolated heteromultimeric Fc-construct of any one of claims 1-17, wherein said heteromultimeric Fc-construct is a bispecific or multispecific antibody.

19. The isolated heteromultimeric Fc-construct of claim 18, comprising at least one antigen-binding domain that binds a cancer antigen.

20. The isolated heteromultimeric Fc-construct of any one of claims 1-17, wherein said heteromultimeric Fc-construct is conjugated to a therapeutic agent.

21. A composition comprising the isolated heteromultimeric Fc-construct of any one of claims 1-20 and a pharmaceutically acceptable carrier.

22. One or more polynucleotides encoding the isolated heteromultimeric Fc-construct of any one of claims 1-19.

23. One or more expression vectors comprising a polynucleotide encoding the isolated heteromultimeric Fc-construct of any one of claims 1-19.

24. A polycistronic expression vector comprising a polynucleotide encoding the isolated heteromultimeric Fc-construct of any one of claims 1-19.

25. A mammalian host cell comprising one or more polynucleotides encoding the isolated heteromultimeric Fc-construct of any one of claims 1-19.

26. A method of expressing the isolated heteromultimeric Fc-construct of any one of claims 1-19 in a mammalian cell, comprising:

a. Transfecting at least one mammalian cell with one or more polynucleotides encoding the isolated heteromultimeric Fc-construct of any one of claims 1-19, thereby producing at least one transiently or stably transfected mammalian cell; and

B. culturing the transiently or stably transfected mammalian cells under conditions suitable for expression of the isolated heteromultimeric Fc construct.

27. Use of the isolated heteromultimeric Fc-construct of claim 19 in the manufacture of a medicament for treating cancer in a patient suffering from cancer characterized by a cancer antigen.